RU2708311C2 - Chimeric antigenic receptors with mnd-promoter - Google Patents

Abstract

FIELD: medicine; biotechnology.SUBSTANCE: invention refers to biotechnology and molecular biology. Disclosed is a lentiviral vector for treating cancer, containing left (5') lentiviral LTR containing a heterologous promoter of cytomegalovirus (CMV), Psi (Ψ) a packing signal, a central polypurine path/DNA-flap (cPPT / FLAP), a rev response element (RRE); (3') lentiviral self-inactivating (SIN) LTR; a synthetic polyadenylation sequence and a polynucleotide comprising a promoter (MND) with a myeloproliferative sarcoma virus enhancer, with a negative-control portion removed, with a primer binding site substituted with a dl587rev sequence operatively linked to a nucleic acid coding a chimeric antigenic receptor (CAR).EFFECT: invention can be used for treating cancer in medicine.12 cl, 9 dwg, 4 tbl, 8 ex

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority in accordance with § 119 (e) of section 35 of the Code of Federal Laws of the United States based on provisional application for US patent No. 61/984561, filed April 25, 2014, which is incorporated herein by reference in its entirety.

STATEMENT OF THE LIST OF SEQUENCES

The sequence listing associated with this application is provided as a file in text format instead of a paper copy and is hereby incorporated by reference into the present description. The name of the text file containing the sequence listing is BLBD_027_01WO_ST25.txt. A text file of 27 kilobytes in size was created on April 24, 2015 and is submitted for review electronically via the EFS-Web simultaneously with the submission of this description.

BACKGROUND OF THE INVENTION

Technical field

The present invention relates to improved compositions and methods for treating cancer or tumor. More specifically, the present invention relates to improved vectors containing chimeric antigenic receptors (CARs), to immune effector cells genetically modified by vectors for expression of such CARs, and to the use of such compositions for the effective treatment of various forms of cancer or tumors.

Description of the Related Art

Cancer is a serious public health problem worldwide. Based on indicators for 2008-2010, 40.76% of men and women who are born today will be diagnosed with some form of cancer at one time or another during their lifetime. In men aged 50 to 70 years, cancer will develop with a probability of 20.37%, and in women with a probability of 15.30%. As of January 1, 2010, there were approximately 13,027,914 live men and women in the United States who had a history of cancer - 6,078,974 men and 6,948,940 women. It is estimated that 1,660,290 men and women (854,790 men and 805,500 women) in the United States will be diagnosed with cancer and 580,350 men and women will die from cancer of various locations in 2013. Howlader et al. 2013.

Despite advances in the detection, prevention and treatment of cancer, a universally successful therapeutic strategy has yet to be implemented. The response of various forms of cancer to treatment is diverse. Traditional treatments for cancer, including chemotherapy and radiation, have limited use due to toxic side effects. Immunotherapy with therapeutic antibodies also provides limited success, partly due to poor pharmacokinetic profiles, rapid elimination of antibodies by serum proteases and glomerular filtration, as well as limited penetration into tumor localization and limited levels of target antigen expression on tumor cells. Attempts to use genetically modified cells expressing chimeric antigenic receptors (CARs) have also been of limited success due to poor in vivo proliferation of T cells with CARs, the rapid disappearance of cells after infusion, and not justified expectations of clinical activity.

Thus, in the art there is a need for more effective compositions and methods for treating cancer from a clinical point of view.

SHORT DESCRIPTION

The present invention generally provides improved vector structures for the production of therapeutic T cells.

In various embodiments, a polynucleotide comprising a promoter (MND) is provided with a myeloproliferative sarcoma virus enhancer, with a deleted negative control region, with a primer binding site substituted for a sequence from dl587rev operably linked to a chimeric antigen receptor (CAR).

In specific embodiments, the CAR contains an extracellular domain that binds an antigen selected from the group consisting of folate receptor alpha, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, Ep , fetal AchR, FRα, GD2, GD3, 'glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO-1, HLA-A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chain, Lewis-Y, kappa chain, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY-ESO -1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM and VEGFR2; a transmembrane domain derived from a polypeptide selected from the group comprising CD8α; CD4, CD28, CD45, PD1 and CD152; one or more intracellular costimulatory signal transmission domains selected from the group consisting of CD28, CD54 (ICAM), CD134 (OX40), CD137 (41BB), CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1) and CD278 (ICOS); and a primary signal transmission domain CD3ζ.

In some embodiments, the extracellular domain comprises an antibody or antigen binding fragment that binds an antigen.

In specific embodiments, an antibody or antigen binding fragment that binds a kappa light chain polypeptide is selected from the group consisting of camel Ig, IgNAR, Fab fragments, Fab'fragments, F (ab) '2 fragments, F (ab ) '3-fragments, Fv, single-chain Fv antibody (“scFv”), bis-scFv, (scFv) 2, minitel, diatel, triatelo, tetra-body, disulfide-stabilized Fv protein (“dsFv”) and single domain antibody ( sdAb, Nanobody).

In further embodiments, the antibody or antigen binding fragment that binds the kappa light chain polypeptide is scFv.

In certain embodiments, the antibody is a human antibody, a murine antibody, or a humanized antibody.

In specific embodiments, the transmembrane domain is derived from CD8α.

In specific embodiments, one or more costimulatory signal transmission domains are selected from the group consisting of CD28, CD134, and CD137.

In some embodiments, a CAR comprises two or more costimulatory signal transmission domains selected from the group consisting of CD28, CD134, and CD137.

In some embodiments, one or more costimulatory signal transmission domains are CD28.

In specific embodiments, one or more costimulatory signal transmission domains are CD134.

In certain embodiments, one or more costimulatory signal transmission domains are CD137.

In specific embodiments, the CAR further comprises a hinge region polypeptide.

In further embodiments, the hinge region polypeptide comprises a hinge region of PD1, CD152, or CD8α.

In further embodiments, the hinge region polypeptide comprises a hinge region of PD1.

In further embodiments, the hinge region polypeptide comprises a hinge region of CD152.

In further embodiments, the hinge region polypeptide comprises a hinge region of CD8α.

In some embodiments, the CAR further comprises a spacer region.

In further embodiments, the spacer region polypeptide comprises CH2 and CH3 regions from IgG1.

In certain embodiments, the CAR further comprises a signal peptide.

In specific embodiments, the signal peptide comprises an IgG1 heavy chain signal polypeptide, a CD8α signal polypeptide, or a human GM-CSF receptor alpha subunit signal peptide.

In some embodiments, a polynucleotide encodes a CAR set forth under any of SEQ ID NO: 2-3.

In various embodiments, a vector is provided comprising a polynucleotide encoding a CAR as contemplated in any of the previous embodiments or in embodiments contemplated elsewhere in this document.

In further embodiments, the vector is an expression vector.

In further embodiments, the vector is a viral vector.

In specific embodiments, the implementation of the vector is a retroviral vector.

In specific embodiments, the implementation of the vector is a lentiviral vector.

In further embodiments, the lentiviral vector is selected from the group consisting essentially of human immunodeficiency virus (HIV); Visna Maedi virus (VMV); goat arthritis encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV) and monkey immunodeficiency virus (SIV).

In certain embodiments, the CAR further comprises a left (5 ') retroviral LTR, a Psi (Ψ) packaging signal, a central polypurine tract / DNA flap (cPPT / FLAP), a retroviral export element; MND promoter operably linked to CAR according to any one of claims. 1-19; and right (3 ') retroviral LTR.

In further embodiments, the CAR further comprises a heterologous polyadenylation sequence.

In further embodiments, the polyadenylation sequence is a bovine growth hormone polyadenylation sequence or rabbit β-globin polyadenylation signal sequence.

In specific embodiments, the CAR further comprises a post-transcriptional regulatory element of hepatitis B virus (HPRE) and a post-transcriptional regulatory element of marmot hepatitis virus (WPRE).

In some embodiments, the 5 ′ LTR promoter is substituted with a heterologous promoter.

In certain embodiments, the heterologous promoter is a cytomegalovirus (CMV) promoter, a Routh sarcoma virus (RSV) promoter, or a monkey 40 virus (SV40) promoter.

In further embodiments, the 5 ′ LTR or 3 ′ LTR is a lentivirus LTR.

In further embodiments, the 3 ′ LTR comprises one or more modifications.

In specific embodiments, the 3 ′ LTR comprises one or more deletions.

In certain embodiments, the 3 'LTR is a self-inactivating (SIN) LTR.

In specific embodiments, a polynucleotide that encodes a CAR comprises an optimized Kozak sequence.

In various embodiments, an immune effector cell is provided comprising a vector described in any of the previous embodiments or in the embodiments described elsewhere in this document.

In some embodiments, the implementation of the immune effector cell is a T-lymphocyte.

In various embodiments, a composition is provided comprising an immune effector cell contemplated in any of the previous embodiments or embodiments contemplated elsewhere in this document, as well as a physiologically acceptable excipient.

In various embodiments, there is provided a method for producing an immune effector cell, comprising introducing into the immune effector cell the vector described herein, stimulating cells and inducing cell proliferation by bringing the cells into contact with antibodies that bind CD3 and antibodies that bind CD28, with thereby obtaining an immune effector cell.

In additional embodiments, the implementation of immune effector cells stimulate and induce proliferation before the introduction of the vector.

In specific embodiments, the implementation of the immune effector cells contain T-lymphocytes.

In various embodiments, there is provided a method for producing an immune effector cell comprising a polynucleotide described herein, comprising isolating CD34 + cells from bone marrow, cord blood, or mobilized peripheral blood cells from a subject and introducing the vector contemplated herein into isolated CD34 + cells.

In further embodiments, the implementation of CD34 + cells is pre-stimulated with one or more cytokines selected from the group comprising the ligand FLT3, TPO, SCF, IL-3 and IL-6, before the introduction of the vector according to any one of paragraphs. 20-36.

In various embodiments, a method is provided for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition contemplated herein.

In certain embodiments, the cancer is selected from the group consisting of Wilms tumor, Ewing's sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, bile duct cancer, cervical cancer, endometrial cancer, esophagus cancer, stomach cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acutemyelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma and bladder cancer.

In specific embodiments, the cancer is pancreatic cancer, and the extracellular binding domain binds the PSCA or MUC1 epitope.

In further embodiments, the cancer is bladder cancer, and the extracellular binding domain binds the PSCA or MUC1 epitope.

In specific embodiments, the cancer is glioblastoma multiforme, and the extracellular binding domain binds an EPHA2, EGFRvIII, or CSPG4 epitope.

In specific embodiments, the cancer is lung cancer, and the extracellular binding domain binds an PSCA or GD2 epitope.

In certain embodiments, the cancer is breast cancer, and the extracellular binding domain binds an epitope of CSPG4 or HER2.

In some embodiments, the cancer is melanoma, and the extracellular binding domain binds an epitope of CSPG4 or GD2.

In various embodiments, a method is provided for treating hemoblastosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition contemplated herein.

In further embodiments, the hemoblastosis is a B-cell neoplasm selected from the group consisting of multiple myeloma (MM), chronic lymphocytic leukemia (CLL), or non-Hodgkin's lymphoma (NHL).

In specific embodiments, the MM is selected from the group consisting of clinically expressed multiple myeloma, sluggish multiple myeloma, plasmacytic leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma and extramedullary plasmacytoma.

In certain embodiments, NHL is selected from the group consisting of Burkitt’s lymphoma, chronic lymphocytic leukemia / small cell lymphocytic lymphoma (CLL / SLL), diffuse B-large cell lymphoma, follicular lymphoma, immunoblastic large-cell lymphoma, lymphoblastic lymphoma from precursor B cells and lymphoma from cells mantle zone.

BRIEF DESCRIPTION OF SEVERAL ASPECTS OF GRAPHIC MATERIALS

Figure 1 shows the structure of the construction of pMND-CD19-CAR (A) and the construction of pMND-kappaLC-CAR (B).

Figure 2 shows a vector map for pMND-CD19-CAR.

Figure 3 shows a vector map for pMND-kappaLC-CAR.

Figure 4 shows the copy number of the vector (VCN) for the integrated lentiviral particles of pMND-kappaLC-CAR. VCN was determined using q-PCR nine days after transduction. Each circle depicts an individual culture obtained in parallel with the corresponding cultures of unmodified T cells (small square). Shows data for 12 individual cultures obtained from 6 donors. The mean and standard deviation are indicated by a line and a "mustache".

Figure 5 shows the expression of kappaLC in T cells transduced with pMND-kappaLC-CAR. The expression of CAR in T cells was determined by flow cytometry 6–9 days after transduction. Each circle depicts an individual culture obtained in parallel with the corresponding cultures of unmodified T cells (small square). Shows data for 12 individual cultures obtained from 6 donors. The mean and standard deviation are indicated by a line and a "mustache".

The figure 6 shows the comparable transduction and expression of CD19-CAR in T cells transduced using lentiviral vectors pMND - or pEF1α-CD19-CAR. These vectors were used to transduce the corresponding parallel cultures of primary human T cells. The expression of CAR in T cells was determined by flow cytometry 6 days after transduction. The number of copies of the vector (VCN) of the integrated lentiviral particles was determined by quantitative PCR nine after transduction.

7 shows tumor specific reactivity in T cells modified with pMND-kappa LC-CAR. The modified T cells were co-cultured with kappa + Daudi cells or HDLM-2 kappa cells for 24 hours. Tumor-specific release of IFN-γ was analyzed by ELISA. Shows data for 5 individual T-cell cultures from 4 donors.

Figure 8 shows the regression of the developed Daudi tumors after adoptive transfer of T cells modified with pMND-kappa LC CAR. Modified T cells were used to treat mice with developing Daudi tumors. Tumor load after treatment was monitored by in vivo imaging compared to untreated control animals. Data are presented for two independent experiments.

The figure 9 shows the antigen-specific clearance of the tumor using T cells expressing CAR. (A). T cells expressing CAR to BCMA led to the cytolysis of BCMA expressing tumor cells labeled with carboxyfluorescein complex succinimidyl ester (CFSE); fluorescence was measured using FACS. (B). T cells expressing CAR for BCMA were co-cultured with K562 cells or K562 cells modified for BCMA expression, and supernatants were harvested after 24 hours and IFN-γ release was analyzed by ELISA. (n = 3).

BRIEF DESCRIPTION OF SEQUENCE IDENTIFIERS

SEQ ID NO: 1 describes a polynucleotide sequence of a promoter (MND) with an enhancer of myeloproliferative sarcoma virus, with a deleted negative control region, with a primer binding site substituted for the sequence from dl587rev.

SEQ ID NO: 2 describes the polynucleotide sequence of a construct containing the MND promoter and CAR to CD19.

SEQ ID NO: 3 sets forth the polynucleotide sequence of a construct containing the MND promoter and Kappa light chain CAR.

DETAILED DESCRIPTION

A. Overview

The present invention generally relates to improved compositions and methods for treating cancer, including without limiting tumors or forms of cancer of the liver, pancreas, lung, breast, bladder, brain, bone, thyroid, kidney, skin, and hematopoietic system. In particular, the present invention relates to adaptive cell therapy with immune effector cells genetically modified using vectors containing a promoter (MND) with an enhancer of the myeloproliferative sarcoma virus, with a deleted negative control region, with a primer binding site, replaced by a sequence from dl587rev, functionally linked with a chimeric antigen receptor.

Genetic approaches offer potential means of enhancing the immune recognition and elimination of cancer cells. One promising strategy is the creation by genetic engineering of immune effector cells for the expression of chimeric antigenic receptors that redirect cytotoxicity to tumor cells. However, existing adaptive cell-based immunotherapy methods for treating tumors or forms of cancer do not have persistent sufficient levels of CAR expression in therapeutic cells. Accordingly, such therapies are not clinically optimal and, therefore, there remains a need in the art for more effective therapies for B-cell neoplasms that would spare humoral immunity.

The improved adaptive cell therapy compositions and methods disclosed herein provide genetically modified immune effector cells that can be easily propagated, which exhibit long-term persistence in vivo and provide stable and sufficient expression of CAR polypeptides. Without wishing to be bound by any particular theory, the present invention considers, in particular, the unexpected observation that the MND promoter controls the stable expression of CAR polypeptides in dormant T cells, activated and propagated, and that such expression is sufficient to efficiently redirect the genetically modified immune effector cells discussed in this document to induce cytotoxic activity against a tumor or cancer cell.

In one embodiment, the polynucleotide comprises an MND promoter operably linked to a polynucleotide encoding a CAR, wherein the CAR comprises an extracellular domain that binds a target antigen, a transmembrane domain, and one or more intracellular signal transduction domains.

In one embodiment, the T cell is genetically modified using a vector containing the MND promoter operably linked to the CAR discussed herein. CAR expressing T cells are referred to herein as CAR-T cells or CAR-modified T cells.

In various embodiments, the genetically modified CAR-T cells described herein are administered to a patient with a cancer or tumor.

In the practice of the present invention, unless otherwise indicated, traditional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology and cell biology, which are within the competence of specialists in this field, will be used, many of which are described below for the purpose of illustration. Such techniques are fully explained in the literature. See, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998) Current Protocols in Immunology Q. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; as well as monographs in magazines such as Advances in Immunology.

All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entireties.

B. Definitions

Unless otherwise specified, all technical and scientific terms used in this document have the same meaning as is well known to ordinary specialists in the field of technology to which the present invention relates. Although any methods and materials similar or equivalent to those described herein can be used in practicing or testing the present invention, preferred embodiments of the compositions, methods and materials are described herein. For the purposes of the present invention, the following terms are defined below.

The singular and plural forms used herein denote one or more (i.e., at least one) grammatical objects of an object. As an example, “element” means one element or several elements.

As used herein, the term “approximately” or “approximately” refers to an amount, level, value, number, frequency, percentage, measurement, size, size, weight, or length that range up to 30, 25, 20, 25, 10 , 9, 8, 7, 6, 5, 4, 3, 2 or 1% relative to the reference amount, level, value, number, frequency, percentage, measurement, size, size, weight, or length. In specific embodiments, the terms “about” or “about”, when used before a numerical value, indicate a plus or minus range of 15%, 10%, 5%, or 1%.

Throughout the present description, unless the context otherwise requires, the words “comprise,” “comprises,” and “comprising” should be understood as implying the inclusion of the indicated stage, or element, or group of stages or elements, but not the exclusion of any other stage, or element, or groups of stages or elements. By “consisting of” is meant the inclusion and limitation of what follows the phrase “consisting of”. Thus, the phrase “consisting of” indicates that the listed items are necessary or required, and that no other items may be present. By “essentially consisting of” is meant to include or contribute to the inclusion of any elements listed after this phrase and restriction to other elements that do not interfere with or contribute to the activities or actions specified in this disclosure in relation to the listed elements. Thus, the phrase “essentially consisting of” indicates that the listed elements are necessary or mandatory, but other elements are optional and may be present or absent depending on whether they affect the activity or action of the listed elements

Reference throughout the present description to “one embodiment”, “embodiment”, “specific embodiment”, “similar embodiment”, “specific embodiment”, “additional embodiment” or “another embodiment” or a combination thereof means that a particular feature, structure, or characteristic described in connection with this embodiment is included in at least one embodiment of the present invention. Thus, the appearance of all of the above phrases in various places throughout the present description does not necessarily apply to one embodiment. Moreover, specific features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

C. Chimeric antigen receptors

In various embodiments, the invention provides immune effector cells obtained by genetic engineering using vectors designed to express chimeric antigenic receptors that redirect cytotoxicity to tumor cells. Such genetically engineered receptors are referred to herein as chimeric antigen receptors (CARs). CARs are molecules that combine the specificity of an antibody to a target antigen (e.g., a tumor antigen) with the intracellular activating domain of a T-cell receptor to produce a chimeric protein that exhibits specific antitumor cellular immune activity. As used herein, the term “chimeric” means composed of portions of various proteins or DNA from various sources.

The vectors discussed herein contain an MND promoter and a polynucleotide encoding CAR. The CARs described herein comprise an extracellular domain that binds to a specific target antigen (also called a binding domain or an antigen-specific binding domain), a transmembrane domain, and an intracellular signal transmission domain. The adhesion of the antigen-binding domain of CAR with its target antigen on the surface of the target cell leads to the formation of clusters from CAR and directs the activation stimulus to CAR-containing cells. The main characteristic of CAR is their ability to redirect the specificity of the immune effector cell, thereby triggering proliferation, cytokine production, phagocytosis or the production of molecules that can mediate cell death of a cell expressing the target antigen in an independent of the main histocompatibility complex (MHC) method, using specific with respect to cell targeting abilities of monoclonal antibodies, soluble ligands, or core-specific coreceptors.

In specific embodiments, the CAR contains an extracellular binding domain including, without limitation, an antibody or antigen binding fragment thereof, a bound ligand or extracellular coreceptor domain that specifically binds a target antigen selected from the group consisting of folate receptor alpha, 5T4, α _v β ₆ - integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, GD2, GD3, 'glypican-3 (GPC3), HLA-A1 + MAGE1, HLA A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO-1, HLA-A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chain, Lewis-Y, kappa chain, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY-ESO -1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM and VEGFR2; one or more hinged domains or spacer domains; transmembrane domain, including without limitation transmembrane domains from CD8α, CD4, CD45, PD1 and CD152; one or more intracellular costimulatory signal transmission domains, including without limitation, intracellular costimulatory signal transmission domains from CD28, CD54 (ICAM), CD134 (OX40), CD137 (41BB), CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1) and CD278 (ICOS); and a primary signal transmission domain of CD3ζ or FcRγ.

1. Binding domain

In specific embodiments, the CARs described herein comprise an extracellular binding domain that specifically binds to a target polypeptide, for example, a target antigen expressed on a tumor cell. As used herein, the terms “binding domain”, “extracellular domain”, “extracellular binding domain”, “antigen-specific binding domain” and “extracellular antigen-specific binding domain” are interchangeable and include CARs with the ability to specifically bind to the target antigen, of interest. The binding domain may contain any protein, polypeptide, oligopeptide or peptide that has the ability to specifically recognize and bind to a biological molecule (e.g., a cell surface receptor or tumor protein, lipid, polysaccharide or other target cell surface molecule or component thereof). A binding domain includes any naturally occurring, synthetic, semisynthetic, or recombinant binding partner for a biological molecule of interest.

The terms “specific binding affinity” or “specifically binding” or “specifically binding” or “specific binding” or “specifically targeting” as used herein describe the binding of one molecule to another with greater binding affinity than background binding . A binding domain (or CAR containing a binding domain, or a fusion protein containing a binding domain) "specifically binds" to the target molecule if it binds or associates with the target molecule with affinity or Ka (that is, the equilibrium association constant of a particular binding interaction in units 1 / M), for example, greater than or equal to approximately 10 ⁵ M ^-1 . In certain embodiments, a binding domain (or a fusion protein based thereon) binds to a target with Ka greater than or equal to about 10 ⁶ M ^-1 , 10 ⁷ M ^-1 , 10 ⁸ M ^-1 , 10 ⁹ M ^-1 , 10 ¹⁰ M ^-1 , 10 ¹¹ M ^-1 , 10 ¹² M ^-1 or 10 ¹³ M ^-1 . Binding domains (or single chain fusion proteins based on them) with “high affinity” are called binding domains with K _a of at least 10 ⁷ M ⁻¹ , at least 10 ⁸ M ⁻¹ , at least 10 ⁹ M ⁻¹ at least 10 ¹⁰ M ^-1 , at least 10 ¹¹ M ^-1 , at least 10 ¹² M ^-1 , at least 10 ¹³ M ^-1 or more.

Alternatively, affinity can be defined as the equilibrium dissociation constant (K _d ) of a particular binding interaction in units of M (for example, from 10 ⁻⁵ M to 10 ⁻¹³ M or less). The affinity of the binding domain polypeptides and CAR proteins according to the present disclosure can be easily determined using conventional techniques, for example, using competitive ELISA (enzyme-linked immunosorbent assay), or binding or substitution association assays using labeled ligands, or using a surface plasmon resonance device such as the Biacore T100, available from Biacore, Inc., Pisktaway, NJ, or optical biosensor technology such as EPIC or EnSpire, available from compan and Corning and Perkin Elmer, respectively (see also, for example, Scatchard et al. (1949) Ann. NY Acad. Sci. 51: 660; U.S. Patent Nos. 5,283,173; 5,468,614 or the like).

In one embodiment, the specific binding affinity is approximately 2 times higher than background binding affinity, approximately 5 times higher than background binding affinity, approximately 10 times higher than background binding affinity, approximately 20 times higher than background binding affinity, about 50 times higher than background affinity binding, approximately 100 times higher than background binding affinity or approximately 1000 times higher than background binding affinity or pain e.

In specific embodiments, the extracellular binding domain of a CAR comprises an antibody or antigen binding fragment thereof. The term “antibody” refers to a binding agent that is a polypeptide containing at least the variable region of an immunoglobulin light chain or heavy chain that specifically recognizes and binds an epitope of an antigen such as a peptide, lipid, polysaccharide or nucleic acid containing an antigenic determinant, recognized by the immune cell.

"Antigen (Ag)" refers to a compound, composition or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions (such as a composition containing a tumor-specific protein) that are injected into an animal or absorbed by an animal. The antigen reacts with products of specific humoral or cellular immunity, including those that were induced by heterologous antigens, such as the disclosed antigens. A “target antigen” or “target antigen of interest” is an antigen for binding of which the CAR binding domain described herein is intended. In specific embodiments, the target antigen is an epitope of a peptide, lipid, polysaccharide or nucleic acid to which a binding domain specifically binds. In preferred embodiments, the antigen is an epitope of the folate receptor polypeptide alpha, 5T4, α _v β ₆ integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FR GD2, GD3, 'glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO-1, HLA-A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chains, Lewis-Y, kappa chains, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSCA , PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGFR2.

An “epitope” or “antigenic determinant” refers to the region of antigen to which a binding agent binds. Epitopes can be formed either from contiguous amino acids or non-contiguous amino acids, brought together by the tertiary structure of the protein. Epitopes formed from contiguous amino acids are typically retained by denaturing solvents, while epitopes formed by a tertiary structure generally disappear upon treatment with denaturing solvents. The epitope typically contains at least 3, often at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.

Antibodies include antigen binding fragments thereof, such as camel Ig, IgNAR, Fab fragments, Fab'fragments, F (ab) '2 fragments, F (ab)' 3 fragments, Fv, single chain Fv proteins (" scFv "), bis-scFv, (scFv) ₂ , minitels, diabodies, triates, tetrabodies, disulfide-stabilized Fv proteins (" dsFv ") and single domain antibodies (sdAb, nanobodies) and parts of the full length antibodies responsible for antigen binding . The term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized mouse antibodies), heteroconjugate antibodies (such as bispecific antibodies), and antigen-binding fragments thereof. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Illinois); Kuby, J., Immunology, 3rd Ed., WH Freeman & Co., New York, 1997.

As will be understood by one of skill in the art and as described elsewhere in this document, a complete antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region and a first, second and third constant region, while each light chain consists of a variable region and a constant region. The mammalian antibody heavy chains are classified as α, δ, ε, γ, and μ, and the mammalian antibody light chains are classified as λ or κ. Immunoglobulins containing heavy chains of α, δ, ε, γ and μ are classified as IgA, IgD, IgE, IgG and IgM immunoglobulins. The complete antibody is Y-shaped. Stem Y consists of the second and third constant regions (and in the case of IgE and IgM, the fourth constant region) of two heavy chains linked together, and disulfide bonds (between chains) are formed in the hinge region. The heavy chains γ, α, and δ contain a constant region consisting of three consecutive (in-line) Ig domains and a hinge region for flexibility; the heavy chains μ and ε contain a constant region consisting of four immunoglobulin domains. The second and third constant regions are called “CH2 domain” and “CH3 domain”, respectively. Each arm Y includes a variable region and a first constant region of one heavy chain associated with the variable and constant regions of one light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

The variable regions of the light and heavy chains comprise a “framework region” interrupted by hypervariable regions, also called “complementarity determining regions” or “CDRs”. CDR can be determined or identified using traditional methods, for example, using the sequence according to Kabat et al. (Wu, TT and Kabat, EA, J Exp Med. 132 (2): 211-50, (1970); Borden, P. and Kabat EA, PNAS 84: 2440-2443 (1987); (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, which is hereby incorporated by reference), or using the structure according to Chothia et al. (Choithia, C. and Lesk, AM, J Mol. Biol., 196 (4): 901-917 (1987), Choithia, C. et al, Nature, 342: 877-883 ( 1989)).

The sequence of frame sections of various light or heavy chains are relatively conservative within a species such as a human. The frame region of the antibody, which is the combined frame regions of the light and heavy chains that make up the antibody, serves to regulate the position and alignment of the CDR in three-dimensional space. First of all, CDRs are responsible for binding to an antigen epitope. The CDRs of each chain are usually called CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, and are also typically identified by the chain in which a particular CDR resides. Thus, CDRs located in the variable domain of the antibody heavy chain are called CDRH1, CDRH2 and CDRH3, while CDRs located in the variable domain of the antibody light chain are called CDRL1, CDRL2 and CDRL3. Antibodies with different specificity (i.e., different paratopes for different antigens) have different CDRs. Although CDRs vary from antibody to antibody, only a limited number of amino acid positions within the CDR are directly involved in antigen binding. Such positions within the CDR are called specificity determining residues (SDR).

References to “V _H ” or “VH” refer to the variable region of an immunoglobulin heavy chain, including that of an antibody, Fv, ScFv, dsFv, Fab, or other antibody fragment disclosed herein. References to “V _L ” or “VL” refer to the variable region of an immunoglobulin light chain, including that of an antibody, Fv, ScFv, dsFv, Fab, or other antibody fragment disclosed herein.

A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or a cell into which the light and heavy chain genes of one antibody have been transfected. Monoclonal antibodies are prepared using methods known to those skilled in the art, for example, by creating antibody-forming hybrid cells by fusion of myeloma cells with spleen immune cells. Monoclonal antibodies include humanized monoclonal antibodies.

A “chimeric antibody” contains framework residues from one species, such as a human, and CDRs (which generally cause antigen binding) from another species, such as a mouse. In specific preferred embodiments, the CARs described herein comprise an antigen-specific binding domain, which is a chimeric antibody or antigen-binding fragment thereof.

In some preferred embodiments, the antibody is a humanized antibody (such as a humanized monoclonal antibody) that specifically binds to a surface protein of a tumor cell. A “humanized” antibody is an immunoglobulin comprising a human framework and one or more CDRs of a non-human immunoglobulin (eg, mouse, rat, or synthetic). The non-human immunoglobulin from which the CDRs originate is called the “donor”, and the human immunoglobulin from which the framework originates is called the “acceptor”. In one embodiment, all CDRs are derived from a donor immunoglobulin in a humanized immunoglobulin. Constant regions may not be present, however, if they exist, they should be almost identical to the constant regions of human immunoglobulins, i.e. be at least about 85-90%, about 95% or more identical. Therefore, all parts of the humanized immunoglobulin, with the possible exception of CDR, are almost identical to the corresponding parts of the sequences of naturally occurring human immunoglobulins. Humanized or other monoclonal antibodies may have additional conservative amino acid substitutions that do not significantly affect antigen binding or other immunoglobulin functions. Humanized antibodies can be constructed using genetic engineering methods (see, for example, US patent No. 5585089).

In specific embodiments, the extracellular binding domain of a CAR comprises an antibody or antigen binding fragment thereof, including, but not limited to, camel Ig (camelid antibody (VHH)), IgNAR, Fab fragments, Fab'-fragments, F (ab) '2- fragments, F (ab) '3-fragments, Fv, single-chain Fv antibody (“scFv”), bis-scFv, (scFv) 2, minitel, diabody, triatelo, tetra-body, stabilized by disulfide bonds Fv-protein (“dsFv” ) and single domain antibody (sdAb, Nanobody).

The “camel Ig” or “camelid VHH antibody” used herein refers to the smallest known antigen-binding unit of an antibody consisting of only heavy chains (Koch-Nolte, et al, FASEB J., 21: 3490-3498 (2007)). The terms “antibody consisting of only heavy chains” or “camelid antibody” refers to an antibody that contains two VH domains and is free of light chains (Riechmann L. et al, J. Immunol. Methods 231: 25–38 (1999) ; WO94 / 04678; WO94 / 25591; US patent No. 6005079).

"IgNAR" or "new immunoglobulin antigen receptor" refers to a class of antibodies from the immune spectrum of sharks, which consist of homodimers from one variable domain of a new antigen receptor (VNAR) and five constant domains of a new antigen receptor (CNAR). IgNARs are one of the smallest known immunoglobulin protein scaffolds, and they are highly stable and have characteristics for efficient binding. Internal stability can be associated with either (i) the underlying Ig framework, which is represented by a significant amount of charged and hydrophilic surface residues, compared to the VH and VL domains of a standard antibody found in murine antibodies; and with (ii) stabilizing structural features in the loops of the complementarity determining regions (CDRs), including disulfide bridges inside the loops and the nature of the hydrogen bonds inside the loops.

Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with one antigen-binding site, and the remaining “Fc” fragment, the name of which reflects its ability to crystallize easily. Pepsin treatment yields an F (ab ') 2 fragment that has two antigen-binding sites and is still capable of cross-linking the antigen.

“Fv” is a minimal antibody fragment that contains a complete antigen binding site. In one embodiment, the double-stranded Fv species consist of a dimer of one heavy chain variable domain and one light chain variable domain in a strong, non-covalent bond. In a single-chain Fv molecule (scFv), one variable domain of the heavy chain and one variable domain of the light chain can be covalently linked via a flexible peptide linker, so that the light and heavy chains can be combined into a "dimeric" structure similar to that of a double-chain Fv molecule. It is in this configuration that the three hypervariable regions (HVRs) of each variable domain interact with the formation of an antigen-binding site on the surface of the VH-VL dimer. Together, these six HVRs determine the antigen binding specificity of the antibody. However, even one variable domain (or half of an Fv containing only three HVRs specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the full binding site.

The Fab fragment contains the variable domains of the heavy and light chains, and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab'fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteine residues from the hinge region of the antibody. Fab'-SH herein refers to Fab ', in which the cysteine residue (s) of the constant domains has a free thiol group. Antibody F (ab ') 2 fragments were originally obtained as pairs of Fab'fragments that share common cysteine residues of the hinge region. Other agents for chemically binding antibody fragments are also known.

The term “diabodies” refers to fragments of an antibody with two antigen-binding sites, the fragments containing the variable domain of the heavy chain (VH) connected to the variable domain of the light chain (VL) in one polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between two domains of the same chain, the domains are forced to pair with the complementary domains of the other chain and create two antigen-binding sites. Diabodies can be bivalent or bispecific. More fully diabodies are described, for example, in EP 404097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., PNAS USA 90: 6444-6448 (1993). Triates and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

“Single domain antibody” or “sdAb” or “nano-body” refers to an antibody fragment that consists of an antibody heavy chain variable region (VH domain) or an antibody light chain variable region (VL domain) (Holt, L., et al, Trends in Biotechnology, 21 (11): 484-490).

"Single chain Fv" or "scFv" antibody fragments contain the VH and VL domains of the antibody, where these domains are present in the same polypeptide chain and in any orientation (for example, VL-VH or VH-VL). Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which allows scFv to form the desired structure for antigen binding. For an overview of scFv, see, for example, Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.

In preferred embodiments, the CAR described herein comprises an antigen-specific binding domain that is scFv and may be murine, human, or humanized scFv. Single chain antibodies can be cloned from genes of the V region of the hybridomas specific for the desired target. Getting such a hybrid has become a tradition. A technique that can be used to clone the variable region of the heavy chain (VH) and the variable region of the light chain (VL) has been described, for example, in Orlandi et al., PNAS, 1989; 86: 3833-3837. In specific embodiments, the antigen-specific binding domain is an scFv that binds a κ or λ light chain polypeptide. In a specific embodiment, scFv binds the folate receptor polypeptide alpha, 5T4, α _v β ₆ integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FR GD3, 'glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO-1, HLA- A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chains, Lewis-Y, kappa chains, mesothelin, Muc1, Muc16, NCAM, ligands NKG2D, NY-ESO-1, PRAME, PSCA, PSMA , ROR1, SSX, survivin, TAG72, TEM or VEGFR2.

An example of a humanized antigen-specific binding domain is an immunoglobulin variable region specific for a tumor antigen that contains at least one human framework site. The term “human framework region” refers to a wild-type framework region (i.e., naturally occurring) of the human immunoglobulin variable region, an altered human immunoglobulin variable region framework region, if less than about 50% (for example, preferably less than about 45%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or 1%) of the amino acids in the site are removed or replaced (for example, by one or more amino acid residues from the frame site of a non-human immunoglobulin, respectively provisions), or an altered framework region of a non-human immunoglobulin variable region if less than about 50% (e.g., less than 45%, 40%, 30%, 25%, 20%, 15%, 10% and 5% ) the amino acids in the region are removed or substituted (for example, at the positions of surface residues and / or one or more amino acid residues from the framework region of the human immunoglobulin at the corresponding positions), thus, in one aspect, the immunogenicity is reduced.

In certain embodiments, the human framework region is a wild-type framework region from the variable region of a human immunoglobulin. In other specific embodiments, the implementation of the human frame region is a modified frame region from the variable region of the human immunoglobulin with amino acid deletions or substitutions in one, two, three, four or five positions. In other embodiments, the human framework region is an altered framework region from a variable region of a non-human immunoglobulin with amino acid deletions or substitutions at one, two, three, four or five positions.

In specific embodiments, the antigen-specific binding domain comprises at least one, two, three, four, five, six, seven, or eight human framework regions (FR) selected from human FR1 light chain, human FR1 heavy chain, human FR2 light chains, human FR2 heavy chain, human FR3 light chain, human FR3 heavy chain, human FR4 light chain and human FR4 heavy chain.

Human FRs that may be present in antigen-specific binding domains also include variants of the FR examples provided herein in which one or two amino acids of the FR examples have been substituted or deleted.

In certain embodiments, the humanized antigen-specific binding domain comprises (a) a humanized light chain variable region that contains a human light chain FR1, human light chain FR2, human light chain FR3 and human light chain FR4, and (b) a humanized heavy variable region a chain that contains the human heavy chain FR1, human heavy chain FR2, human heavy chain FR3, and human heavy chain FR4.

The antigen-specific binding domains provided herein also contain one, two, three, four, five or six CDRs. Such CDRs may be non-human CDRs or modified non-human CDRs selected from CDRL1, CDRL2 and CDRL3 of the light chain and CDRH1, CDRH2 and CDRH3 of the heavy chain. In certain embodiments, the antigen-specific binding domain comprises (a) a light chain variable region that contains light chain CDRL1, light chain CDRL2 and light chain CDRL3, and (b) a heavy chain variable region that contains heavy chain CDRH1, heavy CDRH2 chains and heavy chain CDRH3.

2. Linkers

In certain embodiments, the CARs described herein may contain linker residues between different domains, for example, between V _H and V _L domains, added to appropriately separate them in space and to ensure conformation of the molecule. CARs described herein may contain one, two, three, four, or five or more linkers. In specific embodiments, the linker is from about 1 to about 25 amino acids, from about 5 to about 20 amino acids, or from about 10 to about 20 amino acids, or any other intermediate stretch of amino acids. In some embodiments, the linker has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25 or more amino acids.

Illustrative examples of linkers include glycine polymers (G) _n ; glycine-serine polymers (G _1-5 S _1-5 ) _n , where n is an integer of at least one, two, three, four or five; glycine-alanine polymers; alanine-serine polymers and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and, therefore, can serve as a neutral binder between the domains of fusion proteins, such as CAR, described herein. Glycine provides significantly greater phi-psi space than alanine and is much less limited than residues with longer chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). One of skill in the art will recognize that the CAR structure in particular embodiments may include linkers that are fully or partially flexible, so that the linker may include a flexible linker as well as one or more parts that cause a less flexible structure to provide the desired CAR structure.

Other examples of linkers include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ ID NO: 4); TGEKP (SEQ ID NO: 5) (see, for example, Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 6) (Pomerantz et al. 1995, supra); (GGGGS) _n where = 1, 2, 3, 4 or 5 (SEQ ID NO: 7) (Kim et al., PNAS 93, 1156-1160 (1996.); EGKSSGSGSESKVD (SEQ ID NO: 8) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: 9) (Bird et al., 1988, Science 242: 423-426), GGRRGGGS (SEQ ID NO : 10); LRQRDGERP (SEQ ID NO: 11); LRQKDGGGSERP (SEQ ID NO: 12); LRQKd (GGGS) ₂ ERP (SEQ ID NO: 13). Alternatively, flexible linkers can be developed using computer-aided design methods a program capable of modeling both DNA-binding sites and peptides per se (Desjarlais & Berg, PNAS 90: 2256-2260 (1993), PNAS 91: 11099-11103 (1994), or using phage display methods.

In specific embodiments, the implementation of CAR contains scFV, which further comprises a linker sequence of the variable region. The "variable region linker sequence" is an amino acid sequence that connects the variable region of the heavy chain with the variable region of the light chain and provides spacer function compatible with the interaction of two binding subdomains, so that the resulting polypeptide retains specific binding affinity for the same target molecule as the antibody which contains the same variable regions of the light and heavy chains. In one embodiment, the variable region linker sequence has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids. In a specific embodiment, the variable region linker sequence comprises a glycine-serine polymer (G _1-5 S _1-5 ) _n , where n is an integer of at least 1, 2, 3, 4 or 5. In another embodiment, the linker the sequence of the variable region contains the amino acid linker (G ₄ S) ₃ .

3. Spacer domain

In specific embodiments, the CAR binding domain is followed by one or more “spacer domains”, which is the region that moves the antigen binding domain away from the surface of the effector cell to ensure proper cell / cell contact, antigen binding, and activation (Patel et al., Gene Therapy , 1999; 6: 412-419). The hinge domain can be obtained from a natural, synthetic, semi-synthetic or recombinant source. In certain embodiments, the spacer domain is a portion of an immunoglobulin comprising, without limitation, one or more constant regions of the heavy chain, for example, CH2 and CH3. The spacer domain may include an amino acid sequence from a naturally occurring hinge region of an immunoglobulin or an altered hinge region of an immunoglobulin.

In one embodiment, the spacer domain comprises CH2 and CH3 from IgG1.

4. Hinged domain

The CAR binding domain is typically followed by one or more “hinge domains” that play a role in regulating the position of the antigen binding domain, moving it away from the surface of the effector cell to ensure proper cell / cell contact, antigen binding, and activation. A CAR typically contains one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain can be obtained from a natural, synthetic, semi-synthetic or recombinant source. The hinge domain may include an amino acid sequence from a naturally occurring hinge region of an immunoglobulin or an altered hinge region of an immunoglobulin.

The term “altered hinge region” refers to (a) a naturally occurring hinge region that contains no more than 30% amino acid changes (for example, up to 25%, 20%, 15%, 10% or 5% of amino acid substitutions or deletions), ( b) parts of a naturally occurring hinge region whose length is at least 10 amino acids (e.g. at least 12, 13, 14 or 15 amino acids), while containing no more than 30% amino acid changes (e.g. up to 25%, 20 %, 15%, 10%, or 5% amino acid substitutions or deletions), or (c) parts of a naturally occurring hinge region ty, which contains the hinge region (the length of which is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or at least 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14 or 15 amino acids). In certain embodiments, one or more cysteine residues in a naturally occurring hinge region of an immunoglobulin may be substituted with one or more other amino acid residues (eg, one or more serine residues). The altered hinge region of the immunoglobulin may alternatively or additionally be characterized in that the proline residue from the hinge region of the wild-type immunoglobulin is replaced by another amino acid residue (e.g., a serine residue).

Other illustrative hinge domains suitable for use in CARs described herein include a hinge region derived from extracellular regions of type 1 membrane proteins, such as CD8α, CD4, CD28, and CD7, which may be wild-type hinge regions of these molecules or may be changed. In another embodiment, the hinge domain comprises a hinge region of CD8α.

5. Transmembrane domain (TM)

A "transmembrane domain" is a part of CAR that combines the extracellular binding part and the intracellular signal transmission domain, and also anchors CAR in the plasma membrane of an immune effector cell. The TM domain can be obtained from a natural, synthetic, semi-synthetic or recombinant source. A TM domain can be obtained from (i.e., contains at least the transmembrane region (s)) of the alpha, beta or zeta chain of the T cell receptor, epsilon-CD3, zeta-CD3, CD4, CD5, CD9, CD 16 , CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, PD-1 and CD154. In a specific embodiment, the TM domain is synthetic and preferably contains hydrophobic residues such as leucine and valine.

In one embodiment, the CAR discussed herein contains a TM domain derived from CD8α. In another embodiment, the CAR described herein comprises a TM domain derived from CD8α and a short oligo or polypeptide linker preferably preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, which binds the TM domain and the intracellular signal transduction domain of CAR. The glycine-serine linker is a particularly suitable linker.

6. Intracellular signal transmission domain

In specific embodiments, the CARs described herein comprise an intracellular signal transduction domain. “Intracellular signal transmission domain” refers to the part of CAR that is involved in transmitting messages on the effective binding of CAR with the target antigen to the interior of the immune effector cell to trigger the effector function of the cell, for example, activation, cytokine production, proliferation and cytotoxic activity, including release cytotoxic factors in the CAR-linked target cell, or other cellular responses caused by antigen binding to the extracellular domain of CAR.

The term "effector function" refers to the specialized function of a cell. The effector function of a T cell, for example, can be a cytolytic activity, or an auxiliary, or an activity including cytokine secretion. Thus, the term “intracellular signal transduction domain” refers to a portion of a protein that transmits a signal to trigger an effector function and which causes a cell to perform a specialized function. Although it is usually possible to use a whole intracellular domain of signal transmission, in many cases it is not necessary to use a whole domain. In cases where a truncated part of the intracellular signal transmission domain is used, such a truncated part can be used instead of the whole domain, provided that it transmits a signal to start the effector function. The term intracellular signal transduction domain is intended to include any truncated portion of the intracellular signal transduction domain sufficient to transmit a signal to trigger an effector function.

It is known that signals generated by TCR alone are not sufficient to fully activate T cells, and that a repeat or costimulatory signal is also required. Thus, it can be said that T-cell activation is mediated by two different classes of intracellular signal transmission domains: primary signal transmission domains that initiate antigen-dependent primary activation by TCR (e.g., TCR / CD3 complex), and costimulatory signal transmission domains that act independent of the antigen, providing a secondary or costimulatory signal. In preferred embodiments, the CAR described herein comprises an intracellular signal transduction domain that comprises one or more “costimulatory signal transduction domains” and “primary signal transduction domain”.

Primary signal transmission domains respond to the primary activation of the TCR complex either by stimulation or by inhibition. Primary signal transduction domains that act in a stimulating manner may contain signal transduction motifs, which are known as immunoreceptor tyrosine activating motifs or ITAM.

Illustrative examples of ITAM-containing primary signal transduction domains that are particularly useful in the present invention include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d. In specific preferred embodiments, the CAR comprises a CD3ζ primary signal transmission domain and one or more costimulatory signal transmission domains. The intracellular domains of primary signal transmission and costimulatory signal transmission can be connected in any order sequentially to the carboxyl end of the transmembrane domain.

The CARs described herein contain one or more costimulatory signal transmission domains to enhance the efficiency and propagation of T cells expressing CAR receptors. As used herein, the term “costimulatory signal transmission domain” or “costimulation domain” refers to the intracellular signal transmission domain from a costimulatory molecule. Costimulatory molecules are cell surface molecules that are not antigenic receptors and Fc receptors that provide the secondary signal required for the effective activation and functioning of T-lymphocytes after binding to the antigen. Illustrative examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), CTLA4, LFA-1, CD2, CD7, LIGHT and NKD2C, and also CD83. In one embodiment, a CAR comprises one or more costimulatory signal transmission domains selected from the group consisting of CD28, CD137, and CD134, as well as a primary CD3ζ signal transmission domain.

In another embodiment, the CAR comprises co-stimulation signal transmission domains CD28 and CD137, as well as a primary CD3ζ signal transmission domain.

In yet another embodiment, the CAR comprises co-stimulation signal transmission domains CD28 and CD134, as well as a primary CD3ζ signal transmission domain.

In one embodiment, the CAR comprises costimulation signal transmission domains CD137 and CD134, as well as a primary CD3ζ signal transmission domain.

In one embodiment, the CAR comprises a CD137 costimulation signal transmission domain, as well as a CD3ζ primary signal transmission domain.

In one embodiment, the CAR comprises a CD134 costimulation signal transmission domain, as well as a primary CD3ζ signal transmission domain.

In one embodiment, the CAR comprises a co-stimulation signal transmission domain CD28, as well as a primary CD3ζ signal transmission domain.

In specific embodiments, the CARs described herein comprise an antibody or antigen binding fragment thereof that specifically binds to a folate receptor polypeptide alpha, 5T4, α _v β ₆ integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2 , EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, GD2, GD3, 'glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO-1, HLA-A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chains, Lewis-Y, kappa chains, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGFR2 expressed on a tumor cell.

In one embodiment, CAR contains scFv that binds the folate receptor polypeptide alpha, 5T4, α _v β ₆ integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAPR, fetal FRα, GD2, GD3, 'glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO- 1, HLA-A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chains, Lewis-Y, kappa chains, mesothelin, Muc1, Muc16, NCAM, ligands NKG2D, NY-ESO-1, PRAME , PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGFR2; a transmembrane domain derived from a polypeptide selected from the group comprising CD8α; CD4, CD45, PD1 and CD152; one or more intracellular costimulatory signal transmission domains selected from the group consisting of CD28, CD54, CD134, CD137, CD152, CD273, CD274 and CD278; and a primary signal transmission domain CD3ζ.

In another embodiment, CAR contains scFv that binds the folate receptor polypeptide alpha, 5T4, α _v β ₆ integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAPR, fetal FRα, GD2, GD3, 'glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO- 1, HLA-A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chains, Lewis-Y, kappa chains, mesothelin, Muc1, Muc16, NCAM, ligands NKG2D, NY-ESO-1, PRAME , PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGFR2; the hinge domain selected from the group comprising the hinge / CH2 / CH3 IgG1 and CD8α, as well as CD8α; a transmembrane domain derived from a polypeptide selected from the group comprising CD8α; CD4, CD45, PD1 and CD152; one or more intracellular costimulatory signal transmission domains selected from the group consisting of CD28, CD134, and CD137; as well as a primary signal transmission domain CD3ζ.

In yet another embodiment, CAR comprises scFv, further comprising a linker that binds a folate receptor polypeptide alpha, 5T4, α _v β ₆ integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33 , CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, Ep FAP, Fetal AchR, FRα, GD2, GD3, Glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO-1, HLA-A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chains, Lewis-Y, kappa chains, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY- ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGFR2; the hinge domain selected from the group comprising the hinge / CH2 / CH3 IgG1 and CD8α, as well as CD8α; a transmembrane domain containing a TM domain obtained from a polypeptide selected from the group comprising CD8α; CD4, CD45, PD1 and CD152, as well as a short oligo- or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length, which binds the TM domain to the intracellular domain of CAR signal transmission ; and one or more intracellular costimulatory signal transmission domains selected from the group consisting of CD28, CD134, and CD137; as well as a primary signal transmission domain CD3ζ.

In a specific embodiment, CAR contains scFv that binds the folate receptor polypeptide alpha, 5T4, α _v β ₆ -integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAPR, fetal FRα, GD2, GD3, 'glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO- 1, HLA-A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chains, Lewis-Y, kappa chains, mesothelin, Muc1, Muc16, NCAM, ligands NKG2D, NY-ESO-1, PRAME , PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGFR2; hinge domain containing a polypeptide of the hinge region of PD1 or CD152; a transmembrane domain of PD1 or CD152 containing a polypeptide linker of approximately 3 amino acids; intracellular CD137 costimulatory signal transmission domain; as well as a primary signal transmission domain CD3ζ.

In a specific embodiment, CAR contains scFv that binds the folate receptor polypeptide alpha, 5T4, α _v β ₆ -integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAPR, fetal FRα, GD2, GD3, 'glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO- 1, HLA-A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chains, Lewis-Y, kappa chains, mesothelin, Muc1, Muc16, NCAM, ligands NKG2D, NY-ESO-1, PRAME , PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGFR2; hinge domain containing a polypeptide of the hinge region of PD1 or CD152; a transmembrane domain of PD1 or CD152 containing a polypeptide linker of approximately 3 amino acids; CD134 costimulatory signal intracellular domain; as well as a primary signal transmission domain CD3ζ.

In a specific embodiment, CAR contains scFv that binds the folate receptor polypeptide alpha, 5T4, α _v β ₆ -integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAPR, fetal FRα, GD2, GD3, 'glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO- 1, HLA-A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chains, Lewis-Y, kappa chains, mesothelin, Muc1, Muc16, NCAM, ligands NKG2D, NY-ESO-1, PRAME , PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGFR2; hinge domain containing a polypeptide of the hinge region of PD1 or CD152; a transmembrane domain of PD1 or CD152 containing a polypeptide linker of approximately 3 amino acids; CD28 costimulatory signal intracellular transmission domain; as well as a primary signal transmission domain CD3ζ.

In a specific embodiment, CAR contains scFv that binds the folate receptor polypeptide alpha, 5T4, α _v β ₆ -integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAPR, fetal FRα, GD2, GD3, 'glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO- 1, HLA-A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chains, Lewis-Y, kappa chains, mesothelin, Muc1, Muc16, NCAM, ligands NKG2D, NY-ESO-1, PRAME , PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGFR2; hinge domain comprising hinge polypeptide / CH2 / CH3 IgG1 and CD8α polypeptide; a CD8α transmembrane domain comprising a polypeptide linker of approximately 3 amino acids; intracellular CD137 costimulatory signal transmission domain; as well as a primary signal transmission domain CD3ζ.

In a specific embodiment, CAR contains scFv that binds the folate receptor polypeptide alpha, 5T4, α _v β ₆ -integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAPR, fetal FRα, GD2, GD3, 'glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO- 1, HLA-A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chains, Lewis-Y, kappa chains, mesothelin, Muc1, Muc16, NCAM, ligands NKG2D, NY-ESO-1, PRAME , PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGFR2; hinge domain containing a CD8α polypeptide; a CD8α transmembrane domain comprising a polypeptide linker of approximately 3 amino acids; CD134 costimulatory signal intracellular domain; as well as a primary signal transmission domain CD3ζ.

In a specific embodiment, CAR contains scFv that binds the folate receptor polypeptide alpha, 5T4, α _v β ₆ -integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAPR, fetal FRα, GD2, GD3, 'glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO- 1, HLA-A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chains, Lewis-Y, kappa chains, mesothelin, Muc1, Muc16, NCAM, ligands NKG2D, NY-ESO-1, PRAME , PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGFR2; hinge domain containing a CD8α polypeptide; a CD8α transmembrane domain comprising a polypeptide linker of approximately 3 amino acids; intracellular domain of transmission of co-stimulating signal CD28; as well as a primary signal transmission domain CD3ζ.

In addition, the structure of the vectors discussed in this document improves the reproduction, long-term persistence and cytotoxic properties of T cells and the persistent expression of CAR throughout the life of the cells compared to unmodified T cells or T cells modified with other vectors.

D. Polypeptides

The present invention provides, in particular, CAR polypeptides or fragments thereof, cells and compositions containing them, as well as vectors that express polypeptides. In preferred embodiments, a polypeptide is provided comprising one or more CARs encoded by the polynucleotide sequence set forth under SEQ ID NO: 2 and 3.

"Polypeptide", "fragment of a polypeptide", "peptide" and "protein" are used interchangeably, unless otherwise indicated, and in accordance with the generally accepted meaning, that is, a sequence of amino acids. The polypeptides are not limited to a specific length, for example, they may contain a full-length protein sequence or a full-length protein fragment, and may include post-translational modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, etc., as well as other modifications known from the prior art , both occurring and not occurring in nature. In various embodiments, the CAR polypeptides described herein comprise a signal (or leader) sequence at the N-terminal end of a protein that co-translates or post-translates the transfer of protein. Illustrative examples of suitable signal sequences (signal peptides) applicable to CAR disclosed herein include, but are not limited to, an IgG1 heavy chain signal polypeptide, a CD8α signal polypeptide, or a human GM-CSF receptor alpha subunit signal peptide. Polypeptides can be prepared using any of a variety of well-known recombination and / or synthesis techniques. The polypeptides disclosed herein specifically encompass CARs of the present disclosure or sequences that have deletions, additions, and / or substitutions of one or more amino acids relative to the CARs disclosed herein.

An “isolated peptide” or “an isolated polypeptide” and the like, as used herein, refers to in vitro isolation and / or purification of a peptide or polypeptide molecule from the cellular environment, as well as from communication with other components of the cell, i.e. it is not significantly associated with in vivo substances. Similarly, the term "isolated cell" refers to a cell that has been obtained from an in vivo tissue or organ and contains virtually no extracellular matrix.

Polypeptides include “polypeptide variants”. Variants of the polypeptide may differ from the naturally occurring polypeptide by one or more substitutions, deletions, additions and / or inserts. Such variants can be found in nature or can be obtained synthetically, for example, by modifying one or more of the above polypeptide sequences. For example, in specific embodiments, it may be necessary to improve the binding affinity and / or other biological properties of CAR by introducing one or more substitutions, deletions, additions and / or inserts into the binding domain, hinge, TM domain, costimulatory signal transmission domain, or transmission domain primary signal of the CAR polypeptide. Preferably, the polypeptides of the present invention include polypeptides characterized by at least about 65%, 70%, 75%, 85%, 90%, 95%, 98% or 99% amino acid identity with them.

Polypeptides include “fragments of a polypeptide”. Fragments of a polypeptide are a polypeptide, which may be monomeric or multimeric, characterized by an amino-terminal deletion, a carboxy-terminal deletion and / or an internal deletion or replacement with respect to a naturally occurring or recombinantly produced polypeptide. In certain embodiments, a polypeptide fragment may comprise an amino acid chain having a length of at least 5 to about 500 amino acids. It should be borne in mind that in certain embodiments, the fragments have a length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids. Particularly suitable fragments of a polypeptide include functional domains, including antigen binding domains or antibody fragments. In the case of an antibody to a kappa or lambda light chain, suitable fragments include, but are not limited to: a CDR region, a heavy or light chain CDR3 region; heavy or light chain variable region; part of an antibody chain or variable region, including two CDRs, etc.

The polypeptide can also be fused in a reading frame or conjugated to a linker or other sequence to facilitate the synthesis, purification or identification of a polypeptide (e.g., poly-His), or to improve the binding of the polypeptide to a solid carrier.

As noted above, the polypeptides of the present invention can be modified in various ways, including characterized by amino acid substitutions, deletions, truncations, and inserts. Methods of such manipulations are well known in the art. For example, amino acid sequence variants of a reference polypeptide can be obtained through DNA mutations. Methods for mutagenesis and nucleotide sequence changes are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al., (1987, Methods in Enzymol, 154: 367-382), US patent No. 4873192, Watson, JD et al., (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987) and the referenced documents cited therein. Recommendations regarding suitable amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.).

In certain embodiments, an embodiment will comprise conservative substitutions. A “conservative substitution” is one in which an amino acid is substituted with another amino acid that has similar properties, whereby a person skilled in the art of peptide chemistry may expect that the secondary structure and the hydropathic nature of the polypeptide will not change. Modifications can be made to the structure of polynucleotides and polypeptides of the present invention, and yet a functional molecule can be obtained that encodes a variant or derivative polypeptide with the necessary characteristics. When it is necessary to change the amino acid sequence of a polypeptide to create an equivalent, or even improved, variant polypeptide of the present invention, a person skilled in the art, for example, can change one or more codons of the coding DNA sequence, for example, in accordance with table 1.

Recommendations for determining which amino acid residues can be replaced, inserted or removed without loss of biological activity can be found using computer programs well known in the art, such as DNASTAR ^™ software. Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions for similarly charged or uncharged amino acids. A conservative amino acid change involves replacement within the same family of amino acids that have similar side chains. Naturally occurring amino acids are usually divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan and tyrosine are sometimes classified together as aromatic amino acids. Suitable conservative substitutions of amino acids in a peptide or protein are known to those skilled in the art and, as a rule, they can be performed without changing the biological activity of the resulting molecule. It will be understood by those skilled in the art that, as a rule, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Examples of conservative substitutions are described in provisional patent application US No. 61/241647, the disclosure of which is incorporated herein by reference.

When making such changes, the hydropathic index of amino acids can be taken into account. The importance of the amino acid hydropathic index in providing consistent biological function of a protein is generally known in the art (Kyte and Doolittle, 1982, incorporated herein by reference). Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). Values are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is known from the prior art that certain amino acids can be replaced by other amino acids with a similar index or index of hydropathicity and nevertheless obtain a protein with similar biological activity, i.e., nevertheless, obtain a protein equivalent in biological function. When making such changes, the replacement of amino acids whose hydropathic indices are within ± 2 is preferred, are within ± 1, are particularly preferred, and are within ± 0.5, are even more preferred. It is also known in the art that the replacement of such amino acids can be effectively performed based on hydrophilicity.

As detailed in US Pat. No. 4,554,101, the following hydrophilicity values were assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (–0.4); proline (–0.5 ± 1); alanine (–0.5); histidine (–0.5); cysteine (-1.0); methionine (-1.3); valine (–1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (–3.4). It should be understood that the amino acid can be replaced by another amino acid characterized by a similar hydrophilicity value and nevertheless to obtain a biologically equivalent, and in particular immunologically equivalent, protein. With such changes, the replacement of amino acids, the hydrophilicity of which is within ± 2, is preferred, within ± 1, is particularly preferred, and within ± 0.5, is even more preferred.

As indicated above, amino acid substitutions can be based on the relative similarity of substituents in the side chain of amino acids, for example, the similarity of their hydrophobicity, hydrophilicity, charge, size, etc.

Polypeptide variants further include glycosylated forms, conjugates formed by aggregation with other molecules, as well as conjugates with the formation of covalent bonds with unrelated chemical fragments (for example, pegylated molecules). Variants with a covalent bond can be obtained by linking functional groups to groups that are in the amino acid chain, or to the N- or C-terminal residue, as is known in the art. Variants also include allelic variants, species variants, and muteins. Truncations or deletions of regions that do not affect the functional activity of proteins also lead to the formation of variants.

In one embodiment, which requires the expression of one or more polypeptides, polynucleotide sequences encoding them can be separated using the IRES sequence, as discussed in other parts of this document. In another embodiment, two or more polypeptides can be expressed as a fusion protein that contains one or more self-cleaving polypeptide sequences.

The polypeptides of the present invention include fusion polypeptides. In preferred embodiments, fusion polypeptides and polynucleotides encoding fusion polypeptides, for example, CAR, are provided. Fusion polypeptides and fusion proteins are called a polypeptide that has at least two, three, four, five, six, seven, eight, nine or ten, or more polypeptide segments. Fusion polypeptides are typically fused at the C-terminus to the N-terminus, although they can also be fused at the C-terminus to the C-terminus, N-terminus to the N-terminus, or N-terminus to the C-terminus. The polypeptides in the fusion protein can be in any order or in a specific order. Fusion polypeptides or fusion proteins may also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences and interspecific homologs, while maintaining the desired transcriptional activity of the fusion polypeptide. Fusion polypeptides can be obtained using chemical synthesis methods or by chemically linking two fragments, or, as a rule, can be obtained using other standard techniques. Ligated DNA sequences encoding a fusion polypeptide are operably linked to appropriate transcriptional or translational control elements, as discussed elsewhere in this document.

In one embodiment, the fusion partner comprises a sequence that promotes protein expression (expression enhancer), resulting in higher yields compared to the native recombinant protein. Other fusion partners can be selected in such a way as to increase the solubility of the protein, or to allow the protein to be directed to the desired intracellular compartments, or to facilitate the transport of fusion protein across the cell membrane.

Fusion polypeptides may further comprise a polypeptide cleavage signal between each of the polypeptide domains described herein. In addition, the cleavage site of the polypeptide can be placed in the sequence of any linker peptide. Examples of peptide cleavage signals include recognition sites for cleaving a polypeptide, such as protease cleavage sites, nuclease cleavage sites (e.g., recognition sites for a rarely cleaved enzyme, recognition sites for self-cleaving ribozymes), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5 (8); 616-26).

Suitable protease cleavage sites and self-cleaving peptides are well known to those skilled in the art (see, for example, Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Examples of protease cleavage sites include, but are not limited to, cleavage sites for Potivirus NIa proteases (e.g., tobacco engraving virus proteases), Potivirus HC proteases, Potivirus P1 (P35) proteases, Bimovirus NIa proteases, RNA-2 encoded bimovirus proteases, Aphthovirus L-protease, Enterovirus 2A protease, Rinovirus 2A protease, Picornavirus 3C protease, Comovirus 24K protease, Nepovirus 24K protease, RTSV 3C-like protease (spherical rice tungro virus), PYVF 3C-like protease (yellow virus parsnip spotting), heparin, thrombin, factor Xa and nterokinazy. In one embodiment, due to the high cleavage accuracy, cleavage sites for the TEV protease (tobacco engraving virus), for example EXXYXQ (G / S) (SEQ ID NO: 14), for example, ENLYFQG (SEQ ID NO: 15) and ENLYFQS ( SEQ ID NO: 16), where X is any amino acid (cleavage by TEV occurs between Q and G or Q and S).

In a specific embodiment, self-cleaving peptides include polypeptide sequences derived from 2A peptides of potivirus and cardiovirus, FMDV (foot and mouth disease virus), type A horse rhinitis virus, Thosea asigna virus and pig tesovirus.

In certain embodiments, a self-cleaving polypeptide site comprises a sequence or domain of a 2A site or 2A-like site (Donnelly et al., 2001. J. Gen. Virol. 82: 1027-1041).

TABLE 2: Examples of 2A sites include the following sequences:

SEQ ID NO: 17 LLNFDLLKLAGDVESNPGP SEQ ID NO: 18 TLNFDLLKLAGDVESNPGP SEQ ID NO: 19 LLKLAGDVESNPGP SEQ ID NO: 20 NFDLLKLAGDVESNPGP SEQ ID NO: 21 QLLNFDLLKLAGDVESNPGP SEQ ID NO: 22 APVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 23 VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT SEQ ID NO: 24 LNFDLLKLAGDVESNPGP SEQ ID NO: 25 LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 26 EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP

In preferred embodiments, the polypeptide described herein comprises a CAR polypeptide.

E. Polynucleotides

In specific embodiments, polynucleotides are provided comprising an MND promoter and a polynucleotide encoding one or more CARs. In preferred embodiments, a polynucleotide comprising an MND promoter operably linked to a polynucleotide encoding one or more CARs set forth under SEQ ID NOs: 2 and 3 is provided. As used herein, the terms “polynucleotide” or “nucleic acid” refer to messenger RNA (mRNA), RNA, genomic RNA (gRNA), plus RNA strands (RNA (+)), RNA minus strands (RNA (-)), genomic DNA (gDNA), complementary DNA (cDNA) or recombinant DNA. Polynucleotides include single and double stranded polynucleotides. Preferably, the polynucleotides of the present invention include polynucleotides or variants characterized by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, 99% or 100% sequence identity with any of the reference sequences described herein (see, for example, List of sequences), while, as a rule, the variant retains at least one biological activity of the reference sequence. In various illustrative embodiments, the implementation of the present invention provides, in particular, polynucleotides containing expression vectors, viral vectors and plasmids-carriers, as well as compositions and cells containing them.

In particular embodiments of the present invention, polynucleotides are provided that encode at least about 5, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 500, 1000, 1250, 1500, 1750 or 2000 or more adjacent amino acid residues of the polypeptide of the present invention, as well as all intermediate length polypeptides. It is easy to understand that “intermediate length” in this context means any length between the given values, for example, 6, 7, 8, 9, etc., 101, 102, 103, etc .; 151, 152, 153, etc. 201, 202, 203, etc.

As used herein, the terms “polynucleotide variant” and “variant”, etc., refer to polynucleotides exhibiting substantial sequence identity with a reference polynucleotide sequence, or polynucleotides that hybridize to a reference sequence under stringent conditions, which are defined later in this document . These terms include polynucleotides in which one or more nucleotides have been added, removed, or substituted with other nucleotides compared to a reference polynucleotide. Moreover, it is well known from the prior art that certain changes, including mutations, additions, deletions and substitutions, can be performed relative to a reference polynucleotide, provided that the modified polynucleotide retains the biological function or activity of the reference polynucleotide.

Enumerations of “sequence identity” or containing, for example, “sequence 50% identical” used herein, refer to the extent to which these sequences are identical when pairwise comparing nucleotides or pairwise comparing amino acids in a comparison window. Thus, the “percentage value of sequence identity” can be calculated by comparing two sequences that are optimally aligned in the comparison window, determining the number of positions in which the identical nucleic acid base (for example, A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) are found in both sequences to obtain the number of matching provisions, dividing the number of matching provisions by the total number of provisions the window of comparison (ie. e. the window size) and multiplying the result by 100 to yield the percentage of sequence identity. There are nucleotides and polynucleotides characterized by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 % or 100% sequence identity with any of the reference sequences described herein, typically provided that the polypeptide variant retains at least one biological activity of the reference polypeptide.

The terms used to describe the relationship of the sequences of two or more polynucleotides or polypeptides include the following: "reference sequence", "comparison window", "sequence identity", "percentage value of sequence identity" and "substantial identity". The length of the "reference sequence" is at least 12, but more often 15-18 and often at least 25 monomer units, including nucleotides and amino acid residues. Since each of the two polynucleotides can contain (1) a sequence (i.e. only part of the complete polynucleotide sequence) that is similar in two polynucleotides, and (2) a sequence that is different in two polynucleotides, comparing the sequence of two (or more) polynucleotides, typically performed by comparing sequences of two polynucleotides in a “comparison window” to identify and compare local regions of similarity of sequences. A “comparison window” refers to a speculative segment of at least 6 adjacent positions, typically from about 50 to about 100, more often from about 100 to about 150, in which the sequence is compared with a reference sequence of the same number of adjacent positions after two the sequences were optimally aligned. The comparison window may contain approximately 20% or fewer additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) to optimally align the two sequences. Optimal sequence alignment for alignment in the comparison window can be done using a computerized implementation of the algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package version 7.0, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA) or by viewing the best alignment (i.e. leading to the highest percentage homology in the comparison window) obtained using any of the various selected methods. You can also mention the family of BLAST programs, for example, disclosed in Altschul et al., 1997, Nucl. Acids Res. 25: 3389. A detailed discussion of sequence analysis can be found in section 19.3 in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15.

As used herein, “isolated polynucleotide” refers to a polynucleotide that has been purified from sequences that flank it in the state encountered in the pyrode, for example, a DNA fragment that has been freed from sequences that are normally located next to the fragment. An “isolated polynucleotide” also refers to complementary DNA (cDNA), recombinant DNA, or another polynucleotide that does not exist in nature and which was created by human hands.

The terms describing the orientation of the polynucleotides include: 5 '(normal end of the polynucleotide with a free phosphate group) and 3' (normal end of the polynucleotide with a free hydroxyl (OH) group). Polynucleotide sequences can be annotated in the 5'-3 'orientation or in the 3'-5 ’orientation. In the case of DNA and mRNA, the 5'-3 'strand is designated as “sense”, “plus” or “coding” strand, since its sequence is identical to the sequence of the matrix RNA precursor (pre-mRNA) [except for uracil (U) in RNA, instead thymine (T) in DNA]. In the case of DNA and mRNA, the complementary strand 3′-5 ′, which is a strand transcribed by RNA polymerase, is designated as a “template”, “antisense”, “minus” or “non-coding” chain. As used herein, the term "reverse orientation" refers to a sequence 5'-3 ’recorded in orientation 3'-5 ', or a sequence 3'-5' recorded in orientation 5'-3 '.

The terms “complementary” and “complementarity” refer to polynucleotides (ie, sequences of nucleotides) that are interconnected according to the rules of base pairing. For example, the complementary strand of the 5 'A G T C A T G 3' DNA sequence is 3 'T C A G T A C 5'. The last sequence is often written backwards complementary with the 5'-end on the left and the 3'-end on the right 5 'C A T G A C T 3'. A sequence that is equivalent to its inverse complementary sequence is considered a palindromic sequence. Complementarity can be "partial", with only some nucleic acid bases coinciding in accordance with the rules for pairing bases. Or there may be “complete” or “total” complementarity between nucleic acids.

Moreover, it will be understood by those skilled in the art that due to the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide, or fragment thereof, or variant, as described herein. Some of these polynucleotides are characterized by minimal homology with the nucleotide sequence of any native gene. However, polynucleotides that differ due to differences in codon usage are specifically addressed in the present invention, for example, polynucleotides that are optimized to select codons that are optimal for humans and / or primates. In addition, alleles of genes containing polynucleotide sequences provided herein can also be used. Alleles are endogenous genes that are altered by one or more mutations, such as deletions, additions and / or substitutions of nucleotides.

The term "nucleic acid cassette", as used herein, refers to genetic sequences within a vector that can express RNA, and then a protein. The nucleic acid cassette contains a promoter and a gene of interest, for example, CAR. The nucleic acid cassette is oriented within the vector in terms of position and sequential placement so that the nucleic acid in the cassette can be transcribed into RNA and, if necessary, translated into a protein or polypeptide that undergoes the corresponding post-translational modifications necessary for activity in the transformed cell, and also moves to the appropriate compartment for biological activity by directing sludge to the corresponding intracellular compartments secretion into the extracellular compartments. Preferably, the 3 ′ and 5 ′ ends of the cassette are adapted for seamless insertion into a vector, for example, it contains restriction endonuclease sites at each end. In a preferred embodiment of the present invention, the nucleic acid cassette comprises the sequence of the MND promoter and chimeric antigen receptor discussed herein. The cassette can be removed and inserted into a plasmid or viral vector as a separate unit.

In specific embodiments, polynucleotides include at least one polynucleotide of interest. As used herein, the term “polynucleotide of interest” refers to a polynucleotide encoding a polypeptide (ie, a polypeptide of interest) inserted into an expression vector that needs to be expressed. The vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polynucleotides of interest. In certain embodiments, a polynucleotide of interest encodes a polypeptide that provides a therapeutic effect in the treatment or prevention of a disease or disorder. The polynucleotides of interest, as well as the polypeptides encoded by them, include both polynucleotides that encode wild-type polypeptides and their functional variants and fragments. In specific embodiments, the functional variant is characterized by at least 80%, at least 90%, at least 95%, or at least 99% identity with the corresponding wild-type reference polynucleotide or polypeptide sequence. In certain embodiments, a functional variant or fragment is characterized by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% biological activity with respect to the corresponding wild-type polypeptide.

In one embodiment, the polynucleotide of interest does not encode the polypeptide, but serves as a template for transcription of miRNA, siRNA or shRNA, ribozyme, or other inhibitory RNAs. In various other embodiments, the polynucleotide comprises a polynucleotide of interest encoding CAR and one or more additional polynucleotides of interest, including, but not limited to, the nucleic acid inhibitory sequence, including without limitation: siRNA, miRNA, shRNA and ribozyme.

As used herein, the terms “siRNA” or “short interfering RNA” refer to a short polynucleotide sequence that mediates the process of sequence-specific post-transcriptional gene silencing, translation inhibition, transcription inhibition, or epigenetic RNAi in animals (Zamore et al., 2000, Cell , 101, 25-33; Fire et al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286, 950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes & Dev., 13, 139-141; and Strauss, 1999, Science, 286, 886). In certain embodiments, siRNA comprises a first strand and a second strand that have the same number of nucleosides; however, the first and second strands are biased so that the two terminal nucleosides on the first and second strands are not paired with the remainder on the complementary chain. In some cases, two such unpaired nucleosides are thymidine residues. siRNA must include a region with a sufficient degree of homology relative to the target gene, and have a sufficient length in nucleotides so that siRNA or its fragment can mediate the downregulation of the target gene. Thus, siRNA includes a region that is at least partially complementary to the target RNA. Complete complementarity between the siRNA and the target is not necessary, but the correspondence between them should be sufficient to ensure that the siRNA or its cleavage product controls sequence-specific silencing, for example, by RNAi-cleavage of the target RNA. Complementarity or degree of homology with the target strand is the most critical parameter for the antisense chain. Despite the fact that full complementarity, in particular the antisense strand, is often required, in some embodiments one or more, but preferably 10, 8, 6, 5, 4, 3, 2 or less mismatches with respect to the target RNA are provided. Mismatches are most permissible in the terminal regions, and if present, they are preferably located in the terminal region or regions, for example, within 6, 5, 4 or 3 nucleotides at the 5 'and / or 3' end. The sense chain should only be sufficiently complementary to the antisense chain to maintain the general two-strand nature of the molecule.

In addition, siRNA can be modified or include nucleoside analogues. The single-stranded regions of siRNA can be modified or include nucleoside analogs, for example, an unpaired region or regions of a hairpin structure, for example, a region that binds two complementary regions, can have modifications or nucleoside analogs. Modifications are also applicable to stabilize one or more 3'- or 5'-ends of siRNA, for example, protection against the action of exonucleases, or to facilitate the introduction of an antisense siRNA agent in RISC. Modifications may include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotide spacers (C3, C6, C9, C12, with nitrogen bases removed, triethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents are phosphoramidites, and which are characterized by another DMT-protected hydroxyl group, which allows multiple binding during RNA synthesis. The length of each siRNA strand may be equal to or less than 30, 25, 24, 23, 22, 21, or 20 nucleotides. The length of the strand is preferably at least 19 nucleotides. For example, the length of each strand may be from 21 to 25 nucleotides. Preferred siRNAs have a duplex region of 17, 18, 19, 20, 21, 22, 23, 24, or 25 pairs of nucleotides, and one or more “sticky ends” of 2-3 nucleotides, preferably one or two 3 ′ “sticky ends” from 2-3 nucleotides.

As used herein, the terms “miRNA” or “miRNA” refer to small non-coding RNAs of 20-22 nucleotides, typically excised from ~ 70 nucleotide precursor structures of self-hybridizing RNA, known as pre-miRNA. miRNAs negatively regulate their targets in one of two ways, depending on the degree of complementarity between miRNA and the target. First, miRNA, which binds with complete or near-complete complementarity to mRNA sequences encoding a protein, induces an RNA-mediated interference pathway (RNAi). miRNAs, which exert their regulatory effect by binding to incompletely complementary sites within the 3'-untranslated regions (UTRs) of their target mRNAs, suppress the expression of target genes post-transcriptionally, apparently at a translation level, via the RISC complex, which similar or possibly identical to that used in the RNAi path. Which is consistent with translation control, miRNAs that use this mechanism lower protein levels of their target genes, but mRNA levels of these genes are only minimally affected. miRNAs encompass both naturally occurring miRNAs and artificially developed miRNAs that can specifically target any mRNA sequence. For example, in one embodiment, one of skill in the art can develop short hairpin RNA constructs expressed as primary miRNA transcripts (e.g., miR-30 or miR-21) of a human. During this development, a processing site is added to the hairpin structure under the influence of Drosha, and, as shown, significantly increase the efficiency of knockdown (Pusch et al., 2004). A hairpin stem consists of 22 dsRNA nucleotides (for example, an antisense strand that is characterized by complete complementarity with the desired target) and a 15-19 nucleotide loop from human miR. The addition of the miR loop and miR30 flanking sequences on one or both sides of the hairpin leads to a more than 10-fold increase in the processing of expressed hairpins under the influence of Drosha and Dicer, when compared with traditional shRNA structures without miRNAs. The increased processing level under the influence of Drosha and Dicer leads to a large production of siRNA / miRNA and greater efficiency of expressed hairpins.

As used herein, the terms “shRNA” or “short hairpin RNA” refers to a double-stranded structure formed by one self-complementary strand of RNA. ShRNA constructs containing a nucleotide sequence identical to the part of either the coding or non-coding sequences of the target gene are preferred for inhibition. It has been found that RNA sequences with insertions, deletions and single point mutations relative to the target sequence can also be effective for inhibition. Preferred is a sequence identity of more than 90%, or even sequence identity of 100% between the inhibitory RNA and a portion of the target gene. In certain preferred embodiments, the length of the duplex-forming part of shRNA is at least 20, 21 or 22 nucleotides, which, for example, corresponds to the size of the RNA products resulting from Dicer-dependent cleavage. In certain embodiments, the length of the shRNA construct is at least 25, 50, 100, 200, 300, or 400 bases. In certain embodiments, the length of the shRNA construct is 400-800 bases. shRNA constructs are highly resistant to changes in loop sequence and loop size.

As used herein, the term “ribozyme” refers to a catalytically active RNA molecule capable of site-specific cleavage of the target mRNA. Several subtypes have been described, for example, ribozymes in the form of a “hammer head” and “hairpin”. The catalytic activity and stability of ribozymes can be improved by replacing deoxyribonucleotides with ribonucleotides in non-catalytic bases. Since ribozymes that cleave mRNA in site-specific recognition sequences can be used to destroy specific mRNAs, the use of a “hammerhead” ribozyme is preferred. Ribozymes in the form of a “hammer head” cleave mRNA at positions determined by flanking regions that form complementary base pairs with the target mRNA. The only requirement is that the target mRNA have the following sequence of two bases: 5′-UG-3 ′. The construction and preparation of ribozymes in the form of a “hammer head” are well known in the art.

A preferred method for delivering a polynucleotide of interest, which is siRNA, miRNA, shRNA, or ribozyme, comprises one or more regulatory sequences, such as, for example, a strong constitutive pol III, for example, the human U6 snRNA promoter, mouse U6 snRNA promoter, H1 promoter Human and mouse RNA and the human tRNA-val promoter, or the strong constitutive pol II promoter, described elsewhere in this document.

The polynucleotides of the present invention, regardless of the length of the coding sequence as such, can be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTR), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, ribosome internal landing sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and self-encoding polynucleotides fusion polypeptides, epitope tags, as described elsewhere in this document or as is known in the art, so that their total length can vary significantly. In this regard, it is believed that a polynucleotide fragment of virtually any length can be used, with the total length being preferably limited by the ease of preparation and use of recombinant DNA in the intended protocol.

Polynucleotides can be obtained, manipulated and / or expressed using any of a variety of well-established techniques known and available in the art. In order to express the desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into the corresponding vector. Examples of vectors are plasmids, autonomously replicating sequences, and mobile genetic elements. Additional examples of vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or artificial chromosome derived from P1 (PAC), bacteriophages such as lambda phage or M13 phage as well as animal viruses. Examples of animal virus categories useful as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g. SV40). Examples of expression vectors are pClneo vectors (Promega) for expression in mammalian cells; pLenti4 / V5-DEST ™, pLenti6 / V5-DEST ™ and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In specific embodiments, the coding sequences of chimeric proteins disclosed herein can be ligated into such expression vectors for expression of the chimeric protein in mammalian cells.

The “control elements” or “regulatory sequences” present in the expression vector are such untranslated regions of the vector — the origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence), introns, polyadenylation sequence , 5 'and 3'-untranslated regions — which interact with host cell proteins to transcribe and translate. Such elements may vary in their strength and specificity. Depending on the vector system and host used, any number of suitable transcription and translation elements can be used, including universal promoters and inducible promoters.

In specific embodiments, a vector for use in practicing the present invention, including but not limited to expression vectors and viral vectors, will include exogenous, endogenous, or heterologous control sequences, such as promoters and / or enhancers. An “endogenous” control sequence is a sequence that is naturally linked to this gene in the genome. An “exogenous” control sequence is a sequence that is placed in close proximity to a gene by manipulation of genes (ie, molecular biology techniques), so that the transcription of this gene is due to the associated enhancer / promoter. A “heterologous” control sequence is an exogenous sequence that originates from a species other than a cell subjected to genetic manipulation.

The term “promoter,” as used herein, refers to a recognition site for a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes polynucleotides functionally linked to the promoter. In specific embodiments, promoters that are active in mammalian cells contain an AT-rich region located about 25-30 bases above the site of transcription initiation, and / or another sequence located 70-80 bases above the start of transcription, the CNCAAT region, where N may be any nucleotide.

The term “enhancer” refers to a segment of DNA that contains sequences capable of providing enhanced transcription and, in some cases, capable of functioning regardless of their orientation relative to another control sequence. An enhancer may function in conjunction or additively with promoters and / or other enhancer elements. The term "promoter / enhancer" refers to a segment of DNA that contains sequences capable of providing both promoter and enhancer functions.

The term "functionally connected" refers to the immediate neighborhood, in which the described components are in a relationship that allows them to function properly. In one embodiment, the term refers to a functional relationship between a nucleic acid expression control sequence (such as a promoter and / or enhancer) and a second polynucleotide sequence, for example, a polynucleotide of interest, where the expression control sequence provides transcription of the nucleic acid corresponding to the second sequence.

As used herein, the term “constitutive expression control sequence” refers to a promoter, enhancer or promoter / enhancer that continuously or continuously provides the ability to transcribe a functionally linked sequence. The constitutive expression control sequence may be a “universal” promoter, enhancer or promoter / enhancer that allows expression in a wide variety of types of cells and tissues, or “specific for a cell”, “specific for a type of cell”, “specific for cell line ”or“ tissue specific ”promoter, enhancer or promoter / enhancer that allows expression in a limited number of cell and tissue types, respectively.

Illustrative universal expression control sequences suitable for use in particular embodiments of the present invention include, but are not limited to, the immediate-early promoter of cytomegalovirus (CMV), the viral promoter of monkey 40 virus (SV40) (e.g., early or late), the murine leukemia virus LTR promoter Moloney (MoMLV), Routh sarcoma virus (RSV) LTR, herpes simplex virus (HSV) promoter (thymidine kinase), H5, P7.5, and smallpox virus P11 promoters, 1-alpha elongation factor promoter (EF1a), factor early marrow response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation initiation factor eukaryotes 4A1 (EIF4A1), 70 kDa heat shock protein 5 (HSPA5), 90 kDa thermal shock beta, representative 1 (HSP90B1), 70 kDa heat shock protein (HSP70), β-kinesin (β-KIN), 26 human ROSA locus (Irions et al., Nature Biotechnology 25, 1477 - 1482 (2007)), promoter ubiquitin C (UBC), phosphoglycerate kinase-1 (PGK) promoter, promoter from cytomegalovirus / chicken β-actin promoter (CAG) promoter, β-actin promoter and promoter (MND) with myeloproliferative sarcoma virus enhancer, remotely a negative control region, with a primer binding site substituted for the sequence from dl587rev (Challita et al., J Virol. 69 (2): 748-55 (1995)).

In a particular embodiment, expression of a polynucleotide containing CAR may be necessary due to a promoter that provides stable and long-term expression of CAR in T cells and at a level sufficient to redirect T cells to cells expressing the target antigen. In a preferred embodiment, the promoter is an MND promoter.

In one embodiment, the vector of the present invention comprises an MND promoter comprising one or more nucleotide inserts, deletions, substitutions or modifications that increase, decrease or stabilize the activity of the MND promoter.

As used herein, “conditional expression” may refer to any type of conditional expression, including without limitation of inducible expression; repressed expression; expression in cells or tissues characterized by a specific physiological, biological or painful condition, etc. This definition is not intended to exclude expression specific for the type of cell or tissue. In certain embodiments of the present invention, conditional expression of a polynucleotide of interest is provided, for example, expression is controlled by exposing the cell, tissue, organism, etc., using a treatment tool or condition that causes expression of the polynucleotide or that causes an increase or decrease in level expressing a polynucleotide encoded by the polynucleotide of interest.

Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters, such as promoters of genes encoding glucocorticoid or estrogen receptors (inducible by treatment with an appropriate hormone), metallothionine promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible interferon), mifepristone-regulated GeneSwitch system (Sirin et al., 2003, Gene, 323: 67), kumate-induced gene switch (WO 2002/088346), tetracycline-dependent regulatory systems stems, etc.

Conditional expression can also be achieved using site-specific DNA recombinase. In accordance with certain embodiments of the present invention, the vector comprises at least one (usually two) site (s) for recombination mediated by site-specific recombinase. As used herein, the terms “recombinase” or “site-specific recombinase” include excision or integrative proteins, enzymes, cofactors, or associated proteins that are involved in a recombination reaction involving one or more recombination sites (eg, two, three, four, five , seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3: 699-707 (1993)) or their mutants, derivatives (e.g., fusion proteins containing recombination protein or fragments thereof), fragments and variants. Illustrative examples of recombinases suitable for use in specific embodiments of the present invention include, without limitation, Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ΦC31, Cin, Tn3-resolvase, TndX, XerC, XerD, TnpX, Hjc , Gin, SpCCE1 and ParA.

Vectors may contain one or more recombination sites for any of a wide variety of site-specific recombinases. It should be understood that the target site for site-specific recombinase is in addition to any site (s) necessary for integrating a vector, for example, a retroviral or lentiviral vector. As used herein, the terms “recombination sequence”, “recombination site” or “site-specific recombination site” refer to a specific nucleic acid sequence that the recombinase recognizes and binds to.

For example, one recombination site for Cre recombinase is loxP, which is a 34 base pair sequence containing two inverted repeats of 13 base pairs (acting as recombinase binding sites) flanking a core sequence of 8 base pairs (see FIG. 1 from Sauer, B., Current Opinion in Biotechnology 5: 521-527 (1994)). Other examples of loxP sites include, but are not limited to, lox511 (Hoess et al., 1996; Bethke and Sauer, 1997), lox5171 (Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al., 2002 ), lox71 (Albert et al., 1995) and lox66 (Albert et al., 1995).

Suitable recognition sites for FLP recombinase include without limitation FRT (McLeod, et al., 1996), F _1, F _2, F ₃ (Schlake and Bode, 1994), F _4, F ₅ (Schlake and Bode, 1994), FRT (LE) (Senecoff et al., 1988), FRT (RE) (Senecoff et al., 1988).

Other examples of recognition sequences are attB, attP, attL and attR sequences, which are recognized by the recombinase enzyme λ-integrase, for example, phi-c31. The φC31 SSR mediates recombination only between the heterotypic sites attB (34 bp long) and attP (39 bp long) (Groth et al., 2000). Each of attB and attP, named because of attachment sites for phage integrase on the bacterial or phage genomes, respectively, contains imperfect inverted repeats that are likely to bind to φC31 homodimers (Groth et al., 2000). Product sites, attL and attR, are virtually immune to further φC31-mediated recombination (Belteki et al., 2003), which makes the reaction irreversible. Regarding the catalysis of the inserts, it was found that the insertion of attB DNA into the attP genomic site is much easier than the insertion of the attP site to the attB genomic site (Thyagarajan et al., 2001; Belteki et al., 2003). Thus, in typical strategies, an attP-carrier “docking site” is located at a specific locus by homologous recombination, which is then cooperated with the attB-carrier input sequence for insertion.

As used herein, an “internal ribosome landing site” or “IRES” refers to an element that facilitates direct internal landing of a ribosome on an initiation codon, such as ATG, cistron (a protein coding region), thereby ensuring cap-independent gene translation. See, for example, Jackson et al., 1990. Trends Biochem Sci 15 (12): 477-83) and Jackson and Kaminski. 1995. RNA 1 (10): 985-1000. In specific embodiments, the vectors contemplated by the present invention include one or more polynucleotides of interest that encode one or more polypeptides. In specific embodiments, to achieve efficient translation of each of the plurality of polypeptides, polynucleotide sequences can be separated using one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides.

As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that greatly facilitates the initial binding of mRNA to the small subunit of the ribosome and enhances translation. The Kozak consensus sequence is (GCC) RCCATGG (SEQ ID NO: 27), where R is purine (A or G) (Kozak, 1986. Cell. 44 (2): 283-92, and Kozak, 1987. Nucleic Acids Res. 15 (20): 8125-48). In specific embodiments, the vectors contemplated by the present invention comprise polynucleotides that contain a Kozak consensus sequence and which encode the desired polypeptide, for example, CAR.

In some embodiments of the present invention, a “suicidal” gene, including an inducible “suicidal” gene, is used in the polynucleotide or cell containing the polynucleotide to reduce the risk of direct toxicity and / or uncontrolled proliferation. In specific aspects, a "suicidal" gene is not immunogenic for a host containing a polynucleotide or cell. A specific example of a “suicidal” gene that can be used is caspase-9, or caspase-8, or cytosine deaminase. Caspase-9 can be activated using a specific chemical dimerization inducer (CID).

In certain embodiments, the vectors contain gene segments that make the immune effector cells of the present invention, for example, T cells, susceptible to in vivo negative selection. By “negative selection” is meant that the introduced cell can be eliminated when the individual's in vivo state changes. A negatively breeding phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negatively selectable genes are known in the art and include, but are not limited to, the following genes: herpes simplex virus type I thymidine kinase gene (HSV-I TK) (Wigler et al., Cell 11: 223, 1977), which confers sensitivity to ganciclovir; cell hypoxanthine phosphoribosyl transferase (HPRT) gene, adenine phosphoribosyl transferase (APRT) gene and bacterial cytosine deaminase gene (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

In some embodiments, genetically modified immune effector cells, such as T cells, comprise a polynucleotide further comprising a positive marker that allows selection of cells with a negatively selectable phenotype in vitro. A positively selectable marker may be a gene that, when introduced into a host cell, expresses a dominant phenotype that allows for positive selection of cells carrying the gene. Genes of this type are well known in the art and include, but are not limited to, the hygromycin B phosphotransferase (hph) gene, which confers hygromycin-B resistance, the aminoglycoside phosphosotransferase (neo or aph) gene from Tn5, which encodes antibiotic resistance G418, the gene dihydrofolate reductase (DHFR), adenosine deaminase (ADA) gene and multidrug resistance gene (MDR).

Preferably, the positively selectable marker and the negatively selectable element are linked, such that the loss of the negatively selectable element is necessarily accompanied by the loss of the positively selectable marker. Even more preferred is the fusion of positive and negative breeding markers, so the loss of one inevitably leads to the loss of the other. An example of a fusion polynucleotide, the expression product of which is a polypeptide that simultaneously gives the required traits for positive and negative selection described above, is a fusion gene of hygromycin phosphotransferase and thymidine kinase (HyTK). Expression of this gene provides a polypeptide that confers resistance to hygromycin B for positive in vitro selection, as well as sensitivity to ganciclovir for negative in vivo selection. See Lupton S. D., et al, Mol. and Cell. Biology 1 1: 3374-3378, 1991. In addition, in preferred embodiments, polynucleotides of the present invention encoding chimeric receptors are in retroviral vectors containing a fusion gene, in particular, conferring hygromycin B resistance for positive in vitro selection, and also ganciclovir sensitivity for in vivo negative selection, for example, in a HyTK retroviral vector described in Lupton, SD et al. (1991), supra. See also PCT Publications US91 / 08442 and PCT / US94 / 05601, from S. D. Lupton, describing the use of bifunctional selectable fusion genes obtained by fusion of dominant positive selectable markers with negative selectable markers.

Preferred positive selectable markers come from genes selected from the group comprising hph, nco and gpt, and preferred negative selectable markers come from genes selected from the group comprising cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT and gpt. Particularly preferred markers are bifunctional breeding fusion genes, with a positive breeding marker derived from hph or neo, and a negative breeding marker derived from cytosine deaminase, or a TK gene or breeding marker.

F. Viral vectors

In specific embodiments, a cell (eg, T cell) is transduced using a retroviral vector, for example, a lentiviral vector encoding CAR. For example, the vector contains an MND promoter and encodes a CAR that combines the antigen-specific binding domain of an antibody that binds the folate receptor polypeptide alpha, 5T4, α _v β ₆ integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP2 , EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, GD2, GD3, 'glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY- ESO-1, HLA-A2 + NY-ESO-1, HLA-A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chains, Lewis-Y, kappa chains, mesothelin, Muc1, Muc16, NCAM, ligands NKG2D, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGF R2, with the intracellular signal transduction domain of CD3ζ, CD28, 4-1BB, Ox40, or any combination thereof. Thus, these transduced T cells can elicit a stable, long-term, and persistent CAR-mediated T cell response.

Retroviruses are a common tool for gene delivery (Miller, 2000, Nature. 357: 455-460). In specific embodiments, a retrovirus is used to deliver a polynucleotide encoding a chimeric antigenic receptor (CAR) to a cell. As used herein, the term “retrovirus” refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy, and then covalently integrates its genomic DNA into the host genome. After the virus integrates into the host genome, it is called a "provirus." The provirus serves as a template for RNA polymerase II and controls the expression of RNA molecules that encode structural proteins and enzymes necessary to produce new viral particles.

Illustrative retroviruses suitable for use in specific embodiments include, but are not limited to: Moloni mouse leukemia virus (M-MuLV), Moloni mouse sarcoma virus (MoMSV), Harvey mouse sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Mouse leukemia virus, Mouse stem cell virus (MSCV), Routh sarcoma virus (RSV) and lentivirus.

As used herein, the term “lentivirus” refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, without limitation: HIV (human immunodeficiency virus; including HIV type 1 and type 2 HIV); Visna Maedi virus (VMV); goat arthritis encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV) and monkey immunodeficiency virus (SIV). In one embodiment, HIV-based vector backbones (i.e., cis-acting elements of the HIV sequence) are preferred. In specific embodiments, the lentivirus is used to deliver to the cell a polynucleotide containing the MND promoter and encoding CAR.

Retroviral vectors and, more specifically, lentiviral vectors can be used in the practice of specific embodiments of the present invention. Accordingly, the term “retrovirus” or “retroviral vector” as used herein is meant to include “lentivirus” and “lentiviral vectors”, respectively.

As used herein, the term “vector” refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. A portable nucleic acid is typically linked to a vector nucleic acid molecule, for example, inserted into it. The vector may contain sequences that control autonomous replication in the cell, or may contain sequences sufficient to allow integration into the DNA of the host cell. Useful vectors include, for example, plasmids (e.g. DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Suitable viral vectors include, for example, defective replication system retroviruses and lentiviruses.

As will be apparent to one of skill in the art, the term “viral vector” is widely used to mean either a nucleic acid molecule (eg, a carrier plasmid) that includes elements derived from a nucleic acid of a virus, which typically facilitate the transfer of a nucleic acid molecule or its integration into the genome of a cell, or a viral particle, which mediates the transfer of nucleic acid. Viral particles will typically include various viral components and sometimes also components of the host cell in addition to nucleic acid (s).

The term "viral vector" can refer to either a virus or a viral particle capable of transferring a nucleic acid into a cell, or to the most transferred nucleic acid. Viral vectors and carrier plasmids contain structural and / or functional genetic elements that originate predominantly from the virus. The term "retroviral vector" refers to a viral vector or plasmid containing structural or functional genetic elements or parts thereof, which are derived primarily from a retrovirus. The term "lentiviral vector" refers to a viral vector or plasmid containing structural or functional genetic elements or parts thereof, including LTR, which are derived primarily from lentivirus. The term "hybrid vector" refers to a vector, LTR or other nucleic acid containing both retroviral, for example, lentiviral, sequences, and non-lentiviral viral sequences. In one embodiment, the hybrid vector refers to a vector or carrier plasmid containing retroviral, e.g. lentiviral, sequences for reverse transcription, replication, integration and / or packaging.

In specific embodiments, the terms “lentiviral vector”, “lentiviral expression vector” can be used to refer to lentiviral carrier plasmids and / or infectious lentiviral particles. If reference is made to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it should be understood that the sequences of such elements are represented in the form of RNA in the lentiviral particles of the present invention and are presented in the form of DNA in DNA plasmids of the present invention.

At each end of the provirus, there are structures called “long terminal repeats” or “LTRs”. The term "long terminal repeat (LTR)" refers to domains of base pairs located at the ends of retroviral DNA, which in the context of their natural sequences are direct repeats and contain regions U3, R and U5. LTRs typically provide functions essential for the expression of retroviral genes (e.g., induction, initiation and polyadenylation of gene transcripts) and for virus replication. LTR contains many regulatory signals, including transcriptional control elements, polyadenylation signals, and the sequences necessary for replication and integration of the viral genome. Viral LTR is divided into three areas called U3, R, and U5. The U3 region contains enhancer and promoter elements. The U5 region is a sequence between the primer binding site and the R region and contains a polyadenylation sequence. The R (repeat) region is flanked by the U3 and U5 regions. LTR consists of the U3, R, and U5 regions and is found at both the 5 ′ and 3 ′ ends of the viral genome. Next to 5 'LTR are sequences necessary for reverse transcription of the genome (tRNA primer binding site) and for efficient packaging of viral RNA into particles (Psi site).

As used herein, the term “packaging signal” or “packaging sequence” refers to sequences located within the retroviral genome that are required for insertion of viral RNA into a viral capsid or particle, see, for example, Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101–2109. Some retroviral vectors use the minimum packaging signal (also called psi [Ψ] sequence) needed to encapsulate the viral genome. Thus, the terms “packaging sequence”, “packaging signal”, “psi” and the symbol “Ψ” used in this document are used with respect to the non-coding sequence necessary for encapsulating retroviral RNA strands during assembly of a viral particle.

In various embodiments, the vectors comprise modified 5 'LTR and / or 3' LTR. Each or both LTRs may contain one or more modifications, including without limitation one or more deletions, insertions, or substitutions. Often, modifications to the 3 'LTR are carried out to increase the security of lentiviral or retroviral systems by imparting replication defects to viruses. As used herein, the term “replication-defective” refers to a virus that is not capable of full-fledged, efficient replication, so that no infectious virions are formed (eg, replication-defective lentiviral offspring). The term "replication competent" refers to a wild-type virus or mutant virus that is capable of replication, so that viral replication of the virus leads to the formation of infectious virions (for example, replication competent lentiviral offspring).

The term “self-inactivating” (SIN) vectors refers to replication-defective vectors, for example, retroviral or lentiviral vectors, in which the right (3 ') LTR enhancer-promoter region, known as the U3 region, has been modified (for example, by deletion or replacement ) to prevent viral transcription after the first cycle of viral replication. This is because, during viral replication, the U3 region of the right (3 ') LTR is used as a template for the U3 region of the left (5') LTR, and thus, the viral transcript cannot be obtained without the U3 promoter promoter. In a further embodiment of the present invention, the 3 'LTR is modified so that the U5 region is substituted, for example, with an ideal poly- (A) sequence. It should be noted that modifications to LTR, such as modifications to 3 'LTR, 5' LTR, or both 3 'and 5' LTR, are also included in the scope of the present invention.

An additional safety enhancement is provided by the substitution of the 5-LTR U3 region with a heterologous promoter to control the transcription of the viral genome during the formation of viral particles. Examples of heterologous promoters that can be used include, for example, the monkey virus 40 (SV40) virus promoter (e.g. early or late), the cytomegalovirus (CMV) promoter (e.g., immediate early), the Moloney murine leukemia virus (MoMLV) promoter the promoter of the Routh sarcoma virus (RSV) and the herpes simplex virus (HSV) promoter (thymidine kinase). Typical promoters are capable of providing high levels of transcription in a Tat-independent manner. This substitution reduces the possibility of recombination to obtain replication-competent viruses, since the complete U3 sequence is not present in the virus production system. In certain embodiments, the heterologous promoter has additional advantages in controlling the manner in which the viral genome is transcribed. For example, a heterologous promoter may be inducible, so that the transcription of all or part of the viral genome will occur only in the presence of inducing factors. Inducing factors include, without limitation, one or more chemical compounds or physiological conditions, such as temperature or pH, under which host cells are cultured.

In some embodiments, the viral vectors comprise a TAR element. The term “TAR” refers to the genetic element of the “transactivation response” located in the R region of lentiviral (eg, HIV) LTR. This element interacts with the lentiviral transactivator (tat) genetic element to enhance viral replication. However, this element is not necessary in embodiments where the U3 region of the 5 'LTR is substituted by a heterologous promoter.

An “R region” refers to a region within a retroviral LTR starting from the beginning of the capping group (ie, the transcription start point) and ending immediately before the start of the poly A pathway. The R region is also defined as flanked by U3 and U5 regions. The R region plays an important role during reverse transcription, ensuring the transfer of a growing DNA strand from one end of the genome to the other.

As used herein, the term “FLAP element” refers to a nucleic acid whose sequence comprises a central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, such as HIV-1 or HIV-2. Suitable FLAP elements are described in US patent No. 6682907 and in Zennou, et al., 2000, Cell, 101: 173. During reverse transcription of HIV-1, the central initiation of a plus DNA strand in the central polypurine tract (cPPT) and central termination in the central termination sequence (CTS) leads to the formation of a three-strand DNA structure: the central DNA flap of HIV-1. Without the desire to be bound by any theory, DNA flap can act as a cis-active determinant of the nuclear import of the lentiviral genome and / or can increase the titer of the virus. In specific embodiments, the retroviral or lentiviral vector backbone contains one or more FLAP elements located in vectors above or below the heterologous genes of interest. For example, in specific embodiments, the carrier plasmid comprises a FLAP element. In one embodiment, the vector of the present invention contains a FLAP element isolated from HIV-1.

In one embodiment, retroviral or lentiviral vector vectors contain one or more export elements. The term "export element" refers to a cis-acting post-transcriptional regulatory element that regulates the transport of an RNA transcript from the nucleus to the cell cytoplasm. Examples of RNA export elements include, but are not limited to, a human immunodeficiency virus (HIV) rev response element (RRE) (see, for example, Cullen et al., 1991. J. Virol. 65: 1053; and Cullen et al., 1991. Cell 58: 423) and the post-transcriptional regulatory element of hepatitis B virus (HPRE). Typically, an RNA export element is located within the 3 'UTR of the gene and can be inserted as one or multiple copies.

In specific embodiments, the expression of heterologous sequences in viral vectors is enhanced by incorporating post-transcriptional regulatory elements, effective polyadenylation sites, and optionally transcription termination signals into the vectors. A variety of post-transcriptional regulatory elements can increase the expression of a heterologous nucleic acid into a protein, for example, the marmot hepatitis virus post-transcriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73: 2886); a post-transcriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5: 3864); and the like (Liu et al., 1995, Genes Dev., 9: 1766). In specific embodiments, the vectors of the present invention comprise a post-transcriptional regulatory element, such as WPRE or HPRE.

In specific embodiments, the vectors of the present invention lack or do not contain a post-transcriptional regulatory element, such as WPRE or HPRE, since in some cases, such elements increase the risk of cell transformation and / or do not significantly or significantly increase the amount of mRNA transcript or do not increase stability mRNA. Therefore, in some embodiments, the vectors of the present invention are devoid or not containing WPRE or HPRE as an additional security measure.

Elements that control the effective termination or polyadenylation of heterologous nucleic acid transcripts increase the expression of heterologous genes. Transcription termination signals are typically below the polyadenylation signal. In specific embodiments, the vectors comprise a polyadenylation sequence at the 3′-end of the polynucleotide encoding the polypeptide to be expressed. As used herein, the term "poly (A) site" or "poly (A) sequence" describes a DNA sequence that controls both the termination and polyadenylation of a growing strand of an RNA transcript using RNA polymerase II. Polyadenylation sequences can increase the stability of mRNA by adding a poly (A) tail to the 3 ′ end of the coding sequence, thereby contributing to an increase in translation efficiency. Effective polyadenylation of the recombinant transcript is necessary because transcripts lacking a poly (A) tail are unstable and easily degrade. Illustrative examples of poly (A) signals that can be used in the vector of the present invention include the ideal poly (A) sequence (e.g., AATAAA, ATTAAA, AGTAAA), poly (A) sequence of bovine growth hormone (BGHpA), a rabbit β-globin poly (A) sequence (rβgpA) or another suitable heterologous or endogenous poly (A) sequence known in the art.

In certain embodiments, the retroviral or lentiviral vector further comprises one or more insulator elements. Insulator elements can help protect the sequences expressed by the lentivirus, for example, therapeutic polypeptides, from the effects of integration sites that can be mediated by cis-acting elements present in genomic DNA and leading to misregistration of the expression of the transferred sequence (i.e., positional effect; see e.g. Burgess-Beusse et al., 2002, Proc. Natl. Acad. Sci., USA 99: 16433; and Zhan et al., 2001, Hum. Genet. 109: 471). In some embodiments, the transfer vectors contain one or more insulator elements in the 3 ′ LTR, and after integrating the provirus into the host genome, the provirus contains one or more insulators in both 5 ′ LTR and 3 ′ LTR due to duplication of 3 ′ LTR. Insulators suitable for use in the present invention include, but are not limited to, a chicken β-globin insulator (see Chung et al., 1993. Cell 74: 505; Chung et al., 1997. PNAS 94: 575; and Bell et al., 1999. Cell 98: 387, incorporated herein by reference). Examples of insulator elements include, without limitation, an insulator from a β-globin locus, such as chicken HS4.

In accordance with certain specific embodiments of the present invention, most or all of the backbone sequences of viral vectors originate from lentivirus, such as HIV-1. However, it should be understood that multiple sources of retroviral and / or lentiviral sequences can be used or combined, and many substitutions and changes can be placed in some lentiviral sequences without impairing the ability of the vector to perform the functions described herein. Moreover, many lentiviral vectors are known in the art, see Naldini et al., (1996a, 1996b and 1998); Zufferey et al., (1997); Dull et al., 1998, US Patent Nos. 6013516; and 5994136, many of which can be adapted to produce the viral vector or carrier plasmid of the present invention.

In various embodiments, the vectors of the present invention comprise a promoter operably linked to a polynucleotide encoding a CAR polypeptide. Vectors can contain one or more LTRs, with each LTR containing one or more modifications, such as one or more nucleotide substitutions, additions or deletions. The vectors may further comprise one or more accessory elements to increase transduction efficiency (e.g., cPPT / FLAP), virus packaging (e.g., Psi (Ψ) packaging signal, RRE), and / or other elements that enhance expression of the therapeutic gene (e.g., poly (A) sequences), and may optionally contain WPRE or HPRE.

In a specific embodiment, the carrier vector of the present invention comprises a left (5 ') retroviral LTR, a central polypurine tract / DNA flap (cPPT / FLAP), a retroviral export element, an MND promoter operably linked to a polynucleotide encoding a CAR polypeptide contemplated in this document, both the right (3 ') retroviral LTR and optionally WPRE or HPRE.

In a specific embodiment, the carrier vector of the present invention comprises a left (5 ') retroviral LTR, a retroviral export element, an MND promoter operably linked to a polynucleotide encoding a CAR polypeptide described herein, and a right (3') retroviral LTR, as well as a poly (A) sequence, and optionally WPRE or HPRE. In another specific embodiment of the present invention, there is provided a lentiviral vector comprising a left (5 ') LTR, cPPT / FLAP, RRE, MND promoter operably linked to a polynucleotide encoding a CAR polypeptide described herein, right (3') LTR and polyadenylation sequence; and optionally WPRE or HPRE.

In a specific embodiment of the present invention, there is provided a lentiviral vector comprising the left (5 ') LTR HIV-1, Psi (Ψ) packaging signal, cPPT / FLAP, RRE, MND promoter operably linked to the polynucleotide encoding the CAR polypeptide described herein document, right (3 ') self-inactivating (SIN) LTR of HIV-1, rabbit β-globin polyadenylation sequence, and optionally WPRE or HPRE.

In another embodiment of the present invention, there is provided a vector comprising at least one LTR, a central polypurine tract / DNA flap (cPPT / FLAP), a retroviral export element, and an MND promoter operably linked to the polynucleotide encoding the CAR polypeptide discussed herein as well as optionally WPRE or HPRE.

In a specific embodiment of the present invention, there is provided a vector comprising at least one LTR, cPPT / FLAP, RRE, MND promoter operably linked to a polynucleotide encoding a CAR polypeptide discussed herein, and a polyadenylation sequence, as well as optionally WPRE or HPRE .

In a specific embodiment of the present invention, at least one HIV-1 SIN LTR, Psi (Ψ) packaging signal, cPPT / FLAP, RRE; An MND promoter operably linked to a polynucleotide encoding a CAR polypeptide described herein and a rabbit β-globin polyadenylation sequence, as well as optionally WPRE or HPRE.

One of ordinary skill in the art will understand that many other various embodiments can be modeled based on existing embodiments of the present invention.

A "host cell" includes cells subjected to transfection, infection, or transduction in vivo, ex vivo or in vitro using the recombinant vector or polynucleotide of the present invention. Host cells may include packaging cells producing cells and cells infected with viral vectors. In specific embodiments, host cells infected with the viral vector of the present invention are administered to a subject in need of therapy. In certain embodiments, the term “target cell” is used interchangeably with the term “host cell” and refers to cells of a desired type transfected, infected, or transduced. In preferred embodiments, the target cell is a T cell.

To obtain an acceptable titer of the virus, large-scale production of viral particles is often necessary. Viral particles are obtained by transfecting a carrier vector into a packaging cell line that contains viral structural and / or auxiliary genes, for example, gag, pol, env, tat, rev, vif, vpr, vpu, vpx or nef genes or other retroviral genes.

As used herein, the term “packaging vector” refers to an expression vector or viral vector that is devoid of a packaging signal and contains a polynucleotide encoding one, two, three, four or more viral structural and / or auxiliary genes. Typically, packaging vectors are included in a packaging cell and are introduced into the cell through transfection, transduction and infection. Methods of transfection, transduction and infection are well known to those skilled in the art. The retroviral / lentiviral carrier vector of the present invention can be introduced into a packaging cell line by transfection, transduction and infection to produce a producing cell or cell line. The packaging vectors of the present invention can be introduced into human cells or cell lines using standard methods, including, for example, transfection using calcium phosphate, lipofection or electroporation. In some embodiments, the packaging vectors are introduced into cells together with a dominant breeding marker, such as neomycin, hygromycin, puromycin, blastocidin, zeocin, thymidine kinase, DHFR, glutamine synthetase or ADA, followed by selection in the presence of an appropriate drug and isolation of clones. A selectable marker gene can be physically linked to genes encoded in a packaging vector, for example, by IRES or self-cleaving viral peptides.

Viral envelope proteins (env) define a range of host cells that can ultimately be infected and transformed with recombinant retroviruses derived from cell lines. In the case of lentiviruses such as HIV-1, HIV-2, SIV, FIV and EIV, env proteins include gp41 and gp120. Preferably, the env viral proteins expressed by the packaging cells of the present invention are encoded in a separate vector from the gag and pol viral genes as previously described.

Illustrative examples of retroviral env genes that can be used in the present invention include, but are not limited to, the sheath MLV, 10A1, BAEV, FeLV-B, RD114, SSAV, Ebola, Sendai virus, FPV (poultry plague virus) genes, and the flu virus. Similarly, genes encoding the envelopes of RNA viruses (e.g., RNA viruses from the families Picornaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridaidaidaeiridaeiridae, Filoviridae, Orthomyxiriridaida as well as DNA viruses (families Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxviridae and Iridoviridae). Typical examples include FeLV, VEE, HFVW, WDSV, SFV, rabies virus, ALV, BIV, BLV, EBV, CAEV, SNV, ChTLV, STLV, MPMV, SMRV, RAV, FuSV, MH2, AEV, AMV, CT10 and EIAV.

In other embodiments, envelope proteins for pseudotyping the virus of the present invention include, without limitation, proteins from any of the following viruses: influenza A virus such as H1N1, H1N2, H3N2 and H5N1 (bird flu), influenza B virus, influenza C virus, hepatitis virus A, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rotavirus, any virus from the group of noroviruses, intestinal adenoviruses, parvovirus, dengue fever virus, monkeypox virus, Mononegavirales viruses, lissavirus such as rabies virus in and Lagos bats mustache, Mokola virus, Duvenhag virus, European bats virus type 1 and 2 and Australian bats virus, ephemerovirus, vesiculovirus, vesicular stomatitis virus (VSV), herpes viruses such as herpes simplex virus type 1 and 2 chickenpox, cytomegalovirus, Epstein-Barr virus (EBV), human herpes virus (HHV), human herpes virus type 6 and 8, human immunodeficiency virus (HIV), human papilloma virus, mouse gamma herpes virus, arenaviruses such as the Argentinean hemorrhagic fever virus in Virus of Bolivian hemorrhagic fever, Sabia virus associated with hemorrhagic fever, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, Lymphocytic choriomeningitis virus (LCMV), Hemorrhagic fever, Congesor with renal syndrome, Rift Valley fever virus, Filoviridae (filovirus), including the Ebola hemorrhagic fever virus and Marburg hemorrhagic fever, Flaviviridae, including kias ursky forest disease, Omsk hemorrhagic fever virus, tick-borne encephalitis virus, and Paramyxoviridae such as Hendra virus and Nipah virus, Variola major and Variola minor viruses (smallpox viruses), alphaviruses such as Venezuelan encephalitis encephalitis horse virus, Eastern Encephalitis virus horses, equine western encephalitis virus, SARS-associated coronavirus (SARS-CoV), West Nile virus and any encephalitis-causing virus.

In one embodiment of the present invention, packaging cells are provided that produce a recombinant retrovirus, for example, a lentivirus pseudotyped with VSV-G glycoprotein.

As used herein, the terms “pseudotyping” or “pseudotyping” refer to a virus whose envelope proteins have been replaced by proteins of another virus having preferred characteristics. For example, HIV can be pseudotyped using envelope proteins, which are the G protein of vesicular stomatitis virus (VSV-G), which allows HIV to infect a wider range of cells, since HIV envelope proteins (encoded by the env gene) normally target the virus on CD4 + cells. In a preferred embodiment of the present invention, lentivirus coat proteins are pseudotyped using VSV-G. In one embodiment of the present invention, packaging cells are provided that produce a recombinant retrovirus, for example, a lentivirus pseudotyped with VSV-G envelope glycoprotein.

As used herein, the term “packaging cell lines” is used to refer to cell lines that do not contain a packaging signal, but stably or transiently express viral structural proteins and replication enzymes (eg, gag, pol and env) that are necessary for proper packaging of viral particles. Any suitable cell line can be used to produce packaging cells of the present invention. Typically, these cells are mammalian cells. In a specific embodiment, the cells used to produce the packaging cell line are human cells. Suitable cell lines that can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC cells 1, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells. In preferred embodiments, the packaging cells are 293 cells, 293T cells, or A549 cells. In another preferred embodiment, the cells are A549 cells.

As used herein, the term “producing cell line” refers to a cell line that is capable of producing recombinant retroviral particles comprising a packaging cell line and a carrier vector construct containing a packaging signal. The production of infectious viral particles and stock solutions of the virus can be performed using traditional techniques. Methods for preparing stock solutions of the virus are known in the art and are illustrated, for example, in Y. Soneoka et al. (1995) Nucl. Acids Res. 23: 628-633, and N. R. Landau et al. (1992) J. Virol. 66: 5110-5113. Infectious viral particles can be collected from packaging cells using conventional techniques. For example, infectious particles can be collected by cell lysis and collection of the cell culture supernatant, as is known in the art. If necessary, the collected viral particles can optionally be purified. Suitable purification techniques are well known to those skilled in the art.

The delivery of a gene (s) or other polynucleotide sequence using a retroviral or lentiviral vector via viral infection rather than transfection is called "transduction". In one embodiment, retroviral vectors are transduced into the cell via infection and provirus integration. In certain embodiments, a target cell, for example, a T cell, is “transduced” if it contains a gene or other polynucleotide sequence delivered to the cell through infection with a viral or retroviral vector. In specific embodiments, the transduced cell contains one or more genes or other polynucleotide sequences delivered to the cell genome using a retroviral or lentiviral vector.

In specific embodiments, host cells transduced with a viral vector of the present invention that expresses one or more polypeptides are administered to a subject to treat and / or prevent B-cell growth. Other methods associated with the use of viral vectors in gene therapy, which can be used in accordance with some variants of implementation of the present invention, are described, for example, in Kay, M. A. (1997) Chest 111 (6 Supp.): 138S-142S; Ferry, N. and Heard, J. M. (1998) Hum. Gene Ther. 9: 1975-81; Shiratory, Y. et al. (1999) Liver 19: 265-74; Oka, K. et al. (2000) Curr. Opin. Lipidol. 11: 179-86; Thule, P. M. and Liu, J. M. (2000) Gene Ther. 7: 1744-52; Yang, N. S. (1992) Crit. Rev. Biotechnol. 12: 335-56; Alt, M. (1995) J. Hepatol. 23: 746-58; Brody, S. L. and Crystal, R. G. (1994) Ann. N.Y. Acad. Sci. 716: 90-101; Strayer, D. S. (1999) Expert Opin. Investig. Drugs 8: 2159-2172; Smith-Arica, J. R. and Bartlett, J. S. (2001) Curr. Cardiol. Rep. 3: 43-49; and Lee, H. C. et al. (2000) Nature 408: 483-8.

G. Genetically Modified Cells

In specific embodiments of the present invention, cells genetically modified for the expression of CARs described herein for use in treating forms of cancer are contemplated. As used herein, the term “genetically engineered” or “genetically modified” refers to the addition of additional genetic material in the form of DNA or RNA to the general genetic material of a cell. The terms “genetically modified cells”, “modified cells” and “redirected cells” are used interchangeably. As used herein, the term “gene therapy” refers to the introduction of additional genetic material in the form of DNA or RNA into the general genetic material of a cell that restores, corrects, or modifies gene expression or serves to express a therapeutic polypeptide, for example, CAR.

In specific embodiments, vectors containing the MND promoter and CAR encoding described herein are introduced and expressed in immune effector cells, thereby redirecting their specificity to the target antigen of interest. An “immune effector cell” is any cell of the immune system that is characterized by one or more effector functions (eg, cytolytic activity of a cytotoxic cell, secretion of cytokines, induction of ADCC and / or CDC).

The immune effector cells of the present invention may be autologous / autologous (“their own”) or non-autologous (“not their own,” for example, allogeneic, syngenic or xenogenic).

“Autologous,” as used herein, refers to cells from the same subject.

“Allogeneic,” as used herein, refers to cells of the same species that are genetically distinct when compared to a given cell.

“Syngenic,” as used herein, refers to cells of another subject that are genetically identical when compared to a given cell.

“Xenogenic,” as used herein, refers to cells of a different species when compared to a given cell. In preferred embodiments, the cells of the present invention are allogeneic.

Illustrative immune effector cells used with the vectors containing CAR discussed herein include T-lymphocytes. The terms “T cell” or “T-lymphocyte” are accepted in the art and are intended to include thymocytes, immature T-lymphocytes, mature T-lymphocytes, resting T-lymphocytes or activated T-lymphocytes. The T cell may be a T-helper cell (Th), for example, a type 1 T-helper cell (Th1) or a type 2 T-helper cell (Th2). A T-cell can be a helper T-cell (HTL; CD4 ⁺ T-cell), CD4 ⁺ T-cell, cytotoxic T-cell (CTL; CD8 ⁺ T-cell), CD4 ⁺ CD8 ⁺ T-cell, CD4 ^- CD8 ^- T cell or any other subpopulation of T cells. Other exemplary T cell populations suitable for use in particular embodiments include naive T cells and memory T cells.

As will be understood by one of ordinary skill in the art, vectors containing the MND promoter and encoding CAR can be introduced into other cells, which can also be used as immune effector cells. In particular, immune effector cells also include NK cells, NKT cells, neutrophils and macrophages. Immune effector cells also include precursors of effector cells, where such precursor cells can be induced to differentiate into immune effector cells in vivo or in vitro. Thus, in specific embodiments, the implementation of an immune effector cell includes precursors of immune effector cells, such as hematopoietic stem cells (HSC), included in a population of CD34 + cells derived from umbilical cord blood, bone marrow or mobilized peripheral blood cells, which after administration to the subject differentiate into mature immune effector cells, or which can be induced in vitro to differentiate into mature immune effector cells.

Used in this document, immune effector cells obtained by genetic engineering to include a vector containing the MND promoter, and encoding antigen-specific CAR, can be called "antigen-specific redirected immune effector cells."

As used herein, the term “CD34 + cell” refers to a cell expressing CD34 protein on its cell surface. As used herein, “CD34” refers to a cell surface glycoprotein (eg, sialomucin protein), which often acts as an intercellular adhesion factor and is involved in the entry of T cells into the lymph nodes. The population of CD34 + cells consists of hematopoietic stem cells (HSC), which, after introduction into the patient, differentiate and participate in the formation of all hematopoietic growths, including T cells, NK cells, NKT cells, neutrophils and monocytic / macrophage cell line cells .

The present invention provides methods for producing immune effector cells that express the CAR described herein. In one embodiment, the method comprises transfection or transduction of immune effector cells isolated from an individual, whereby the immune effector cells express one or more CARs described herein. In certain embodiments, immune effector cells are isolated from the individual and genetically modified without further in vitro manipulation. Such cells can then be directly re-administered to the individual. In further embodiments, the immune effector cells are first activated and stimulated to proliferate in vitro before being genetically modified to express CAR. In this regard, immune effector cells can be cultured before and / or after they have undergone genetic modification (i.e., they are transduced or transfected for the expression of CAR discussed in this document).

In specific embodiments, prior to in vitro manipulation or genetic modification of the immune effector cells described herein, a cell source is obtained from a subject. In specific embodiments, the implementation of CAR-modified immune effector cells contain T cells. T cells can be obtained from various sources, including without limitation mononuclear cells in peripheral blood, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue and tumors. In certain embodiments, T cells can be obtained from a dose of blood collected from a subject using any of a variety of techniques known to a person skilled in the art, for example, by sedimentation, for example, separation using FICOLL ^™ . In one embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nuclear white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in a suitable buffer or medium for subsequent processing. Cells can be washed with PBS or another suitable solution that does not contain calcium, magnesium, and most, if not all other divalent cations. As will be appreciated by those of ordinary skill in the art, the washing step can be performed using methods known to those skilled in the art, such as a semi-automatic flow centrifuge. For example, Cobe 2991 Cell Processor, Baxter CytoMate, etc. After washing, cells can be resuspended in various biocompatible buffers or other saline solutions containing or not containing buffer. In certain embodiments, unwanted components from a sample after apheresis can be removed in culture medium to directly resuspend cells.

In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMC) by lysis of red blood cells and depletion of monocytes, for example, by centrifugation in a PERCOLL ^™ gradient. Specific subpopulations of T cells expressing one or more of the following markers: CD3, CD28, CD4, CD8, CD45RA and CD45RO can be further isolated using positive or negative selection techniques. In one embodiment, a specific subpopulation of T cells expressing CD3, CD28, CD4, CD8, CD45RA, and CD45RO is further isolated using positive or negative selection techniques. For example, enrichment of the T-cell population through negative selection can be accomplished using a combination of antibodies aimed at surface markers characteristic of negatively breeding cells. One method for use herein is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, using a mixture of monoclonal antibodies directed to cell surface markers present for negatively selectable cells. For example, to enrich for CD4 ⁺ cells by negative selection, a mixture of monoclonal antibodies typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present invention.

PBMC can be targeted genetically modified using vectors containing the MND promoter that is in functional communication for the expression of the polynucleotide encoding the CAR discussed in this document. In certain embodiments, after isolation of PBMCs, T lymphocytes are further isolated and in some embodiments, both cytotoxic and helper T lymphocytes can be sorted into subpopulations of naive, memory, and effector T cells either before or after genetic modification and / or breeding.

CD8 ⁺ cells can be obtained using standard methods. In some embodiments, CD8 ⁺ cells are further sorted into naive, central memory cells and effector cells by identifying cell surface antigens that are associated with each of these types of CD8 ⁺ cells.

In certain embodiments, naive CD8 ⁺ T lymphocytes are characterized by the expression of phenotypic markers of naive T cells, including CD62L, CCR7, CD28, CD3, CD 127 and CD45RA.

In specific embodiments, memory T cells are present in subpopulations of both CD62L ⁺ and CD62L ^- CD8 ⁺ peripheral blood lymphocytes. PBMCs are sorted into CD62L ^- CD8 ⁺ and CD62L ⁺ CD8 ⁺ fractions after staining with anti-CD8 and CD62L antibodies. In some embodiments, phenotypic markers CD45RO, CD62L, CCR7, CD28, CD3, and CD127 are expressed on central memory T cells, and they are negative for granzyme B. In some embodiments, central memory T cells are CD45RO ⁺ , CD62L ⁺ , CD8 ⁺ T cells.

In some embodiments, effector T cells are negative for CD62L, CCR7, CD28 and CD127, and positive for granzyme B and perforin.

In certain embodiments, CD4 ⁺ T cells are further sorted into subpopulations. For example, CD4 ⁺ T helper cells can be sorted into naive, central memory cells and effector cells by identifying cell populations that have cell surface antigens. CD4 ⁺ lymphocytes can be obtained using standard methods. In some embodiments, naive CD4 ⁺ T lymphocytes are CD45RO ^- , CD45RA ⁺ , CD62L ⁺ CD4 ⁺ T cells. In some embodiments, central CD4 ⁺ memory cells are CD62L-positive and CD45RO-positive. In some embodiments, effector CD4 ⁺ cells are CD62L and CD45RO negative.

Immune effector cells, such as T cells, can be genetically modified, followed by isolation using standard methods, or immune effector cells can be activated and expanded (or differentiated in the case of progenitor cells) in vitro before undergoing genetic modification. In a specific embodiment, immune effector cells, such as T cells, are genetically modified using the chimeric antigen receptors discussed herein (for example, transduced using a viral vector containing the MND promoter and a nucleic acid encoding CAR) and then activate and propagated in vitro. In various embodiments, T cells can be activated and propagated before or after genetic modification for CAR expression using methods described, for example, in US Pat. Nos. 6352694; 6,534,055; 6905680; 6692964; 5,858,358; 6,887,466; 6905681; 7144575; 7,067,318; 7172869; 7,232,566; 7,175,843; 5,883,223; 6905874; 6797514; 6867041 and in the publication of patent application US No. 20060121005.

Typically, T cells are propagated by contacting a surface characterized by an agent attached to it that stimulates the signal associated with the CD3-TCR complex and a ligand that stimulates a co-stimulating molecule on the surface of T cells. T cell populations can be stimulated by contacting an anti-CD3 antibody or antigen binding fragment thereof, or an anti-CD2 antibody immobilized on the surface, or by contacting a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. Costimulation of auxiliary molecules on the surface of T cells is also considered.

In specific embodiments, PBMCs or isolated T cells are contacted with a stimulant and a costimulatory agent, such as anti-CD3 and CD28 antibodies, typically attached to a granule or other surface, in a culture medium with appropriate cytokines such as IL- 2, IL-7 and / or IL-15. To stimulate the proliferation of either CD4 ⁺ T cells or CD8 ⁺ T cells, an anti-CD3 antibody and an anti-CD28 antibody can be used, and other methods well known in the art can be used (Berg et al., Transplant Proc. 30 (8): 3975-3977, 1998; Haanen et al., J. Exp. Med. 190 (9): 13191328, 1999; Garland et al., J. Immunol Meth. 227 (1 -2): 53-63 , 1999). Examples of antibodies to CD28 include 9.3, B-T3, XR-CD28 (Diacione, Besancon, France). An anti-CD3 antibody and an anti-CD28 antibody attached to a single granule act as a "surrogate" antigen presenting cell (APC). In other embodiments, T cells can be activated and stimulated to proliferate using feeder cells, as well as appropriate antibodies and cytokines, by methods such as described in US6040177; US5827642 and WO2012129514.

In other embodiments, artificial APCs (aAPCs) are obtained by developing K562, U937, 721.221, T2, and C1R cells that are designed to stably express and secrete a variety of costimulatory molecules and cytokines. In a particular embodiment, aAPCs in the form of K32 or U32 are used to expose one or more antibody-based stimulatory molecules on the AAPC cell surface. Expression of various gene combinations on aAPC provides the ability to accurately determine the requirements for activation of human T cells, so that aAPC can be adapted to optimally multiply subpopulations of T cells with specific requirements for the growth medium and having different functions. aAPCs support ex vivo growth and long-term reproduction of functional human CD8 T cells that do not require the addition of exogenous cytokines, in contrast to the use of natural APCs. T cell populations can be propagated by aAPC expressing various costimulatory molecules, including but not limited to CD137L (4-1BBL), CD134L (OX40L) and / or CD80 or CD86. Finally, aAPCs provide an effective platform for the propagation of genetically modified T cells and for maintaining the expression of CD28 on CD8 T cells. The aAPCs provided in WO 03/057171 and US2003 / 0147869 are hereby incorporated by reference in their entirety.

In one embodiment, CD34 ⁺ cells are transduced using the nucleic acid construct of the present invention. In certain embodiments, transduced CD34 ⁺ cells differentiate into mature immune effector cells in vivo after administration to a subject, typically a subject, from which these cells were originally isolated. In another embodiment, CD34 ⁺ cells can be stimulated in vitro before exposure or after they are genetically modified by CAR, as described herein, using one or more of the following cytokines: Flt-3 ligand (FLT3), stem factor cells (SCF), growth factor and differentiation of megakaryocytes (TPO), IL-3 and IL-6 in accordance with the methods described previously (Asheuer et al., 2004; Imren, et al., 2004).

The present invention provides a population of modified immune effector cells for the treatment of cancer, wherein the modified immune effector cells contain the CARs disclosed herein. For example, a population of modified immune effector cells is obtained from peripheral blood mononuclear cells (PBMCs) obtained from a patient with the diagnosed B-cell neoplasm described herein (autologous donors). PBMCs form a heterogeneous population of T-lymphocytes, which may be CD4 +, CD8 + or CD4 + and CD8 +.

PBMCs may also include other cytotoxic lymphocytes, such as NK cells or NKT cells. An expression vector containing a promoter, for example, an MND promoter, and the coding sequence of the CAR discussed herein can be introduced into a population of human donor T cells, NK cells, or NKT cells. Successfully transduced T cells that carry an expression vector can be sorted using flow cytometry to isolate CD3-positive T cells and then further expanded to increase the number of these T cells expressing the CAR protein, in addition to activating cells using antibodies to CD3 and / or antibodies to CD28 and IL-2 or any other methods known from the prior art described in other parts of this document. Standard procedures are used to cryopreserve T cells expressing a CAR protein, to store T cells and / or a preparation for use in a human subject. In one embodiment, in vitro transduction, cultivation and / or propagation of T cells is carried out in the absence of non-human animal products, such as fetal calf serum and fetal bovine serum. Since the heterogeneous population of PBMC is genetically modified, the resulting transduced cells are a heterogeneous population of modified cells containing the antigen-specific targeted CAR discussed in this document.

In a further embodiment, when genetically modifying a donor population of immune effector cells, a mixture of, for example, one, two, three, four, five or more different expression vectors can be used, where each vector encodes a protein of a different chimeric antigen receptor, as discussed herein. The resulting modified immune effector cells form a mixed population of modified cells, with a certain fraction of the modified cells expressing several different CAR proteins.

In one embodiment, the present invention provides a method for storing genetically modified immune effector cells expressing a murine, human, or humanized CAR protein, comprising cryopreservation of immune effector cells, such that the cells remain viable after thawing. The fraction of immune effector cells expressing CAR proteins can be cryopreserved using methods known in the art to provide a constant source of such cells for future treatment of cancer patients. If necessary, cryopreserved transformed immune effector cells can be thawed, grown and propagated to increase the number of such cells.

As used herein, “cryopreservation” refers to the preservation of cells by cooling to temperatures below zero, such as (typically) 77 K or −196 ° C. (boiling point of liquid nitrogen). In order to prevent the destruction of stored cells during freezing at low temperatures or during heating to room temperature, cryoprotective agents are often used. Cryoprotective agents and optimal cooling rates can protect cells from damage. Cryoprotective agents that can be used include, but are not limited to, dimethyl sulfoxide (DMSO) (Lovelock and Bishop, Nature, 1959; 183: 1394-1395; Ashwood-Smith, Nature, 1961; 190: 1204-1205), glycerin, polyvinylpyrrolidine (Rinfret, Ann. NY Acad. Sci., 1960; 85: 576) and polyethylene glycol (Sloviter and Ravdin, Nature, 1962; 196: 48). The preferred cooling rate is 1 ° -3 ° C / minute. After at least two hours, T cells reach a temperature of −80 ° C and can be placed directly in liquid nitrogen (−196 ° C) for permanent storage, for example, in a cryogenic vessel for long-term storage.

H. Compositions and compositions

The compositions contemplated herein may contain one or more polypeptides, polynucleotides, vectors containing them, genetically modified immune effector cells, etc., as contemplated herein. Compositions include, without limitation, pharmaceutical compositions. “Pharmaceutical composition” refers to a composition formulated in pharmaceutically acceptable or physiologically acceptable solutions for administration to a cell or animal, either alone or in combination with one or more other therapies. It should also be borne in mind that, if desired, the compositions of the present invention can also be administered in combination with other agents, such as, for example, cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies or other various pharmaceutically active agents. For other components, which can also be included in the composition, there are practically no restrictions, provided that additional funds do not adversely affect the ability of the composition to deliver the intended therapeutic agent.

The phrase "pharmaceutically acceptable" is used in this document to refer to those compounds, materials, compositions and / or finished dosage forms that, in the context of medical indications, are suitable for use in contact with human and animal tissues, while not causing excessive toxicity, irritation , an allergic reaction, or other problem or complication commensurate with an acceptable benefit / risk ratio.

As used herein, a “pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, an adjuvant, carrier, excipient, lubricant, sweetener, diluent, preservative, colorant / coloring agent, flavor and odor enhancer, surfactant, wetting agent, dispersing agent agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant or emulsifier that has been approved by the Quality Control Authority US Food and Drug Administration as suitable for use in humans or pets. Examples of pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol; phosphate buffers and any other compatible substances used in pharmaceutical formulations.

In specific embodiments, the compositions of the present invention comprise a number of genetically modified immune effector cells, as described herein. As used herein, the term “amount” refers to an “amount effective” or “effective amount” of a genetically modified therapeutic cell, for example, T cells, to achieve a useful or desired prophylactic or therapeutic result, including clinical results.

A “prophylactically effective amount” refers to an amount of a genetically modified therapeutic cell effective to achieve the desired prophylactic result. Usually, but not necessarily, since a prophylactic dose is used in subjects before the onset of the disease or at its early stage, the prophylactically effective amount is less than the therapeutically effective amount.

The “therapeutically effective amount” of a genetically modified therapeutic cell can vary depending on factors such as the stage of the disease, the age, gender and weight of the individual, as well as the ability of stem and progenitor cells to elicit the desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or harmful effects of the virus or transduced therapeutic cells are outweighed by therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective for “treating” a subject (eg, a patient). If a therapeutic amount is indicated, the exact number of compositions of the present invention to be administered can be determined by the attending physician taking into account individual differences in age, weight, tumor size, degree of infection or metastases, and the condition of the patient (subject). In general, it can be indicated that the pharmaceutical composition containing the T cells described herein can be administered at a dose of 10 ² -10 ¹⁰ cells / kg body weight, preferably 10 ⁵ -10 ⁶ cells / kg body weight, including all integer values within these ranges. The number of cells will depend on the end use method for which the composition is intended, as well as on the type of cells included in it. For the methods of use provided herein, the number of cells per liter of volume or less may be 500 ml or less, even 250 ml or 100 ml or less. Therefore, the density of the required cells typically exceeds 10 ⁶ cells / ml and usually exceeds 10 ⁷ cells / ml, typically 10 ⁸ cells / ml or more. A clinically significant number of immune cells can be divided into several infusions that are collectively equal to or greater than 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ or 10 ¹² cells. In some aspects of the present invention, in particular, if all of the introduced cells are redirected to a specific target antigen (for example, κ or λ light chains), reduced cell numbers in the range of 10 ⁶ / kg (10 ⁶ -10 ¹¹ per patient ) Compositions based on cells expressing CAR can be administered several times at dosages within the indicated ranges. Cells can be allogeneic, syngeneic, xenogenic or autologous to the patient undergoing treatment. Optionally, to enhance the induction of an immune response, treatment may also include the administration of mitogens (e.g., PHA) or lymphokines, cytokines and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-alpha, IL-18 and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) described herein.

Typically, compositions containing cells activated and propagated as described herein can be used in the treatment and prevention of diseases that occur in immunocompromised individuals. In particular, compositions comprising CAR-modified T cells discussed herein are used in the treatment of cancer. The CAR-modified T cells of the present invention can be administered either singly or as a pharmaceutical composition in combination with carriers, diluents, excipients and / or other components, such as IL-2 or other cytokines or cell populations. In specific embodiments, the pharmaceutical compositions described herein comprise a number of genetically modified T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

The pharmaceutical compositions of the present invention containing a population of CAR-expressing immune effector cells, such as T cells, may contain buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins polypeptides or amino acids, such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g. aluminum hydroxide) and preservatives. The compositions of the present invention are preferably formulated for parenteral administration, for example, intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.

Liquid pharmaceutical compositions, whether solutions, suspensions or other similar forms, may include one or more of the following: sterile diluents such as water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride solution, fixed oils, such as synthetic mono- or diglycerides, which can serve as a solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and tonicity agents such as sodium chloride or dextrose. A preparation for parenteral administration can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. The injectable pharmaceutical composition is preferably sterile.

In a specific embodiment, the compositions described herein comprise an effective amount of CAR-expressing immune effector cells alone or in combination with one or more therapeutic agents. Thus, compositions based on CAR-expressing immune effector cells can be administered alone or in combination with other known cancer treatments such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. Compositions can also be administered in combination with antibiotics. Such therapeutic agents can be accepted in the art as standard treatment for a particular disease condition described herein, such as a particular form of cancer. Examples of therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory drugs, chemotherapeutic agents, radiotherapeutic agents, therapeutic antibodies, or other active and auxiliary agents.

In certain embodiments, compositions comprising CAR-expressing immune effector cells disclosed herein can be administered in combination with any number of chemotherapeutic agents. Illustrative examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN ™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboxwon, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide and trimethylolomelamine resin; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichine, phenesterol, prednimustine, trophosphamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomisins, actinomycin, anthramycin, azaserin, bleomycin, cactinomycin, calichemicin, carabicin, carminomycin, carcinophilin, chromomycin, dactinomycin, daunorubicin, 6-diazoinocinoricin-dicoricinoricin-5-diazoinubicorinbicorin idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimerexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, karmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine, phloxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostan, testolactone; agents that inhibit the function of the adrenal cortex, such as aminoglutetimide, mitotane, trilostane; a folic acid supplement, such as folinic acid; Aceglaton; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisanthrene; edatrexate; defofamine; demecolcin; diaziquon; elformitin; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pyrarubicin; podophyllic acid; 2-ethyl hydrazide; procarbazine; PSK®; razoxane; sisofiran; spiro germanium; tenuazonic acid; triaziquon; 2, 2 ', 2' '- trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacitosin; arabinoside ("Ara-C"); cyclophosphamide; thiotepu; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (TAXOTERE®., Rhne-Poulenc Rorer, Anthony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; Navelbin; novantron; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomitin (DMFO); retinoic acid derivatives such as Targretin ™ (bexarotene), Panretin ™ (alitretinoin); ONTAK ™ (Denileukin Diftitox); esperamycins; capecitabine; and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing. Also included in this definition are anti-hormonal agents that regulate or inhibit the action of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, 4 (5) -imidazoles, aromatase inhibitors, 4-hydroxy tamoxifen, trioxifene, keoxifen, LY117018, onapriston and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

A variety of other therapeutic agents can be used in combination with the compositions described herein. In one embodiment, a composition comprising CAR-expressing immune effector cells is administered with an anti-inflammatory agent. Anti-inflammatory drugs or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), non-steroidal anti-inflammatory drugs (tsuprofen, i-aspirin, i naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide and mycophenolate.

Other examples of NSAIDs are selected from the group consisting of ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as VIOXX® (rofecoxib) and CELEBREX® (celecoxib) and sialylates. Examples of pain relievers are selected from the group consisting of acetaminophen, oxycodone, tramadol or propoxyphene hydrochloride. Examples of glucocorticoids are selected from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone or prednisone. Examples of biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE) ®)), chemokine inhibitors and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies, as well as recombinant forms of molecules. Examples of DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, a gold preparation (oral (auranofin) and intramuscular) and minocycline.

Illustrative examples of therapeutic antibodies suitable for combination with the CAR-modified T cells discussed herein include, but are not limited to, Abagovab, Adecatumumab, Afutuzumab, Alemtuzumab, Altumab, Amatuximab, Anatumomab, Artsitumab, Bavituximab, Bectumomab, Bevatsumab brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab, tsiksutumab, klivatuzumab, conatumumab, daratumumab, drositumab, duligotumab, dusigitumab, detumomab, datsetuzumab, dalotuzumab, ercomex b, elotuzumab, ensituksimab, ertumaksomab, etaratsizumab, farietuzumab, fiklatuzumab, figitumumab, flanvotumab, futuksimab, ganitumab, gemtuzumab, girentuksimab, glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuksimab, inotuzumab, intetumumab, ipilimumab, iratumumab, labetuzumab, leksatumumab, lintuzumab, lorvotuzumab, lukatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, moxetumomab, narnatumab, naptumab, necitumumab, nimotuzumab, nofetumumab, okaratuzumab, ofatumumab, onartuma , Oregovomab, panitumumab, parsatuzumab, partitumab, pemtumomab, pertuzumab, pintumomab, pritumumab, rakotumomab, radretumab, rilotumumab, rituximab, robatumumab, satumomab, sibrotuzumab, siltuksimab, simtuzumab, solitomab, takatuzumab, taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab , tukotuzumab, ublituximab, veltuzumab, vorsetuzumab, voteumab, zalutumumab, CC49 and 3F8.

In certain embodiments, the compositions described herein are administered in combination with a cytokine. By “cytokine” as used herein is meant a generic term for proteins released by one population of cells that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and cattle growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and luteinizing hormone (LH); hepatocyte growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor alpha and beta; Mueller inhibitory substance; murine gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet growth factor; transforming growth factors (TGF), such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon alpha, beta and gamma; colony stimulating factors (CSF) such as macrophage-CSF (M-CSF); granulocyte macrophage-CSF (GM-CSF) and granulocyte-CSF (G-CSF); interleukins (IL), such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL-11, IL-12; IL-15, a tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term “cytokine” includes proteins from natural sources or from a culture of recombinant cells, as well as biologically active equivalents of cytokines with a native sequence.

I. Target cells and antigens

The present invention provides, in particular, genetically modified immune effector cells redirected to a target cell, for example, a tumor or cancer cell, and which contain CAR with a binding domain that binds to target antigens on the cells. As used herein, the term “cancer” refers generally to a class of diseases or conditions in which abnormal cells divide uncontrollably and are able to invade nearby tissues. Cancer cells can also spread to other parts of the body through the circulatory and lymphatic systems. There are several main types of cancer. A carcinoma is a cancer that begins in the skin or in the tissues lining or covering the internal organs. Sarcoma is a cancer that begins in bone, cartilage, adipose tissue, muscle tissue, blood vessels, or other connective or supporting tissue. Leukemia is a cancer that begins in the hematopoietic tissue, such as bone marrow, and causes the appearance of a large number of abnormal blood cells and their entry into the blood. Lymphoma and multiple myeloma are forms of cancer that begin in the cells of the immune system. Cancer forms of the central nervous system are forms of cancer that begin in the tissues of the brain and spinal cord.

As used herein, the term "malignant" refers to cancer in which a group of tumor cells exhibits one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., invasion and destruction of adjacent tissues), and metastasis (i.e., spreading to other parts of the body through lymph or blood). As used herein, the term “metastasize” refers to the spread of cancer from one part of the body to another. A tumor formed by spreading cells is called a “metastatic tumor” or “metastasis”. A metastatic tumor consists of cells similar to the cells of the original (primary) tumor.

As used herein, the term “benign” or “non-malignant” refers to tumors that can grow in size but do not spread to other parts of the body. Benign tumors are self-limiting and usually are not prone to invasion or metastasis.

“Cancer cell” or “tumor cell” refers to a single cell of a cancerous tumor or tissue. A tumor, in general, is called bloating or a pathological change formed due to abnormal cell growth, which can be benign, precancerous or malignant. Most forms of cancer form tumors, but some, such as leukemia, do not necessarily form tumors. For those forms of cancer that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably. The amount of a tumor in an individual is a “tumor burden,” which can be measured as the number, volume, or weight of the tumor.

In one embodiment, the target cell expresses an antigen, for example, the target antigen, which is practically not found on the surface of other normal (required) cells. In one embodiment, the target cell is a pancreatic parenchyma cell, pancreatic duct cell, liver cell, myocardial cell, skeletal muscle cell, osteoblast, skeletal myoblast, neuron, vascular endothelial cell, pigment cell, smooth muscle cell, glial cell, fat cell , osteocyte, chondrocyte, pancreatic islet cell, CNS cell, PNS cell, liver cell, lipocyte, kidney cell, lung cell, skin cell, ovarian cell, follicular cell, epithelial cell immune cell or endothelial cell.

In certain embodiments, the target cell is a portion of pancreatic tissue, nerve tissue, heart tissue, bone marrow, muscle tissue, bone tissue, skin tissue, liver tissue, hair follicle, vascular tissue, adipose tissue, lung tissue, and kidney tissue.

In a specific embodiment, the target cell is a tumor cell. In another specific embodiment, the target cell is a cancer cell, such as a cell in a patient with cancer. Examples of cells that can be destroyed using the disclosed methods include cells of the following tumors: hemoblastosis, such as leukemia, including acute leukemia (such as acute lymphocytic leukemia, acute myelocytic leukemia, and myeloblast, promyelocytic, myelomonocytic, monocytic and erythroleukemia) chronic leukemia (such as chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia), true polycythemia, lymphoma, Hodgkin's disease, non-Hodgkin lymphoma, multiple myeloma, m Waldenstrom acroglobulinemia, heavy chain disease).

In another embodiment, the cell is a solid tumor cell, such as a form of sarcoma or carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma and other sarcomas, synovioma, mesothelioma, Ewing sarcoma, colon carcinoma, papilloma leiomyosarcoma, , breast cancer, ovarian cancer, prostate cancer, liver cell cancer, lung cancer, colorectal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (e.g. adenocarcinoma pancreas, colon, ovary, lung, breast, stomach, prostate, cervix or esophagus), carcinoma of the sweat gland, carcinoma of the sebaceous gland, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic cancer, renal cell carcinoma , bile duct carcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumor, bladder carcinoma, CNS tumors (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblast ma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma).

In one embodiment, the cancer is selected from the group consisting of Wilms tumor, Ewing's sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, colon cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, bile duct cancer, cervical cancer, endometrial cancer, esophagus cancer, stomach cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myelogen leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma and bladder cancer.

In one embodiment, the target cell is a malignant cell of the liver, pancreas, lung, breast, bladder, brain, bone, thyroid, kidney, skin, and hematopoietic system. In another embodiment, the target cell is a cell in liver cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, brain cancer, bone cancer, thyroid cancer, kidney cancer, skin cancer or hematopoietic cancer.

In one embodiment, the target antigen is an folate receptor polypeptide epitope of alpha, 5T4, α _v β ₆ integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR , GD2, GD3, Glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO-1 , HLA-A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chains, Lewis-Y, kappa chains, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGFR2.

J. Therapies

The genetically modified T cells discussed herein provide improved adoptive immunotherapy methods for use in treating various tumors and forms of cancer. In specific embodiments, the specificity of the primary T cell is redirected to tumor or cancer cells by genetic modification of the primary T cell using the CAR described herein. In various embodiments, the viral vector is used to genetically modify an immune effector cell with a polynucleotide containing an MND promoter and encoding a CAR containing an antigen-specific binding domain that binds a folate receptor polypeptide alpha, 5T4, α _v β ₆ integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, in including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, GD2, GD3, 'glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO-1, HLA-A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chains, Lewis-Y, kappa chains, mesothelin, Muc1, Muc16, NCAM, ligands NKG2D, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72 , TEM or VEGFR2; hinge domain; a transmembrane domain containing a TM domain derived from a polypeptide selected from the group comprising CD8α; CD4, CD45, PD1 and CD152, as well as a short oligo- or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length, which binds the TM domain to the intracellular domain of CAR signal transmission ; and one or more intracellular costimulatory signal transmission domains selected from the group consisting of CD28, CD134, and CD137; as well as a primary signal transmission domain CD3ζ.

In one embodiment, the present invention provides a type of cell therapy in which T cells are genetically modified to express CAR, which targets cancer cells expressing a target antigen, and CAR-T cells are administered to a recipient in need thereof. The introduced cells are able to destroy the tumor cells in the recipient. Unlike antibody-based therapy, CAR-T cells are able to replicate in vivo, leading to long-term persistence, which can provide long-term cancer therapy.

In one embodiment, the CAR-T cells of the present invention can undergo active in vivo propagation of T cells and can persist for an extended period of time. In another embodiment, the CAR-T cells of the present invention are converted to specific memory T cells that can be reactivated to inhibit any additional tumor formation or growth.

In specific embodiments, compositions comprising an immune effector cell genetically modified with a vector containing an MND promoter operably linked to a polynucleotide encoding CAR are used in the treatment of solid tumors or cancers, including without limitation of liver cancer, pancreatic cancer lung cancer, breast cancer, bladder cancer, brain cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer.

In specific embodiments, compositions comprising an immune effector cell genetically modified using a vector containing an MND promoter operably linked to a polynucleotide encoding a CAR that contains an antigen-specific binding domain that binds an PSCA or MUC1 epitope are used in the treatment of pancreatic cancer glands.

In specific embodiments, compositions comprising an immune effector cell genetically modified with a vector comprising an MND promoter operably linked to a polynucleotide encoding a CAR that contains an antigen-specific binding domain that binds an EPHA2, EGFRvIII or CSPG4 epitope are used in the treatment of glioblastoma multiforme.

In specific embodiments, compositions comprising an immune effector cell genetically modified using a vector containing an MND promoter operably linked to a polynucleotide encoding a CAR that contains an antigen-specific binding domain that binds an PSCA or MUC1 epitope are used in the treatment of urinary cancer the bubble.

In specific embodiments, compositions comprising an immune effector cell genetically modified with a vector containing an MND promoter operably linked to a polynucleotide encoding a CAR that contains an antigen-specific binding domain that binds an PSCA or GD2 epitope are used in the treatment of lung cancer .

In specific embodiments, compositions comprising an immune effector cell genetically modified with a vector containing an MND promoter operably linked to a polynucleotide encoding a CAR that contains an antigen-specific binding domain that binds an CSPG4 or HER2 epitope are used in the treatment of breast cancer glands, for example, thrice-negative breast cancer.

In specific embodiments, compositions comprising an immune effector cell genetically modified with a vector containing an MND promoter operably linked to a polynucleotide encoding a CAR that contains an antigen-specific binding domain that binds an GD2 or CSPG4 epitope are used in the treatment of melanoma.

In specific embodiments, compositions comprising an immune effector cell genetically modified using a vector containing an MND promoter operably linked to a polynucleotide encoding a CAR are used in the treatment of hemoblastoses, including without limitation of leukemia, including acute leukemia (e.g. ALL, AML and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemia (e.g. CLL, SLL, CML, HCL), true polycythemia, lymphoma, Hodgkin’s disease, non-Hodgkin’s lymphoma , multiple myeloma, Waldenstrom macroglobulinemia and heavy chain disease.

In specific embodiments, compositions comprising an immune effector cell genetically modified using a vector containing an MND promoter operably linked to a polynucleotide encoding CAR are used in the treatment of B-cell tumors, including without limitation of multiple myeloma (MM), non-Hodgkin’s lymphomas (NHL) and chronic lymphocytic leukemia (CLL).

Multiple myeloma is a B-cell neoplasm with the morphology of mature plasma cells, characterized by malignant degeneration of one clone of these cell types. These plasma cells proliferate in BM and can invade adjacent bone and sometimes blood. Variant forms of multiple myeloma include clinically expressed multiple myeloma, sluggish multiple myeloma, plasmacytic leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma and extramedullary plasmacytoma (see, for example, Braunwalds et al.). of Internal Medicine, 15th Edition (McGraw-Hill 2001)).

Non-Hodgkin's lymphoma covers a large group of forms of cancer of lymphocytes (white blood cells). Non-Hodgkin lymphomas can occur at any age and are often characterized by enlarged lymph nodes, fever and weight loss. There are many types of non-Hodgkin lymphoma. For example, non-Hodgkin's lymphoma can be divided into aggressive (fast-growing) and sluggish (slow-growing) types. Although non-Hodgkin’s lymphomas can be derived from B cells and T cells, the terms “non-Hodgkin’s lymphoma” and “B-cell non-Hodgkin’s lymphoma” are used interchangeably. Non-Hodgkin’s B-cell lymphomas (NHL) include Burkitt’s lymphoma, chronic lymphocytic leukemia / small cell lymphocytic lymphoma (CLL / SLL), diffuse B-large cell lymphoma, follicular lymphoma, immunoblastic large-cell lymphoma, lymphoblastic lymphoma from progenitor B cell and lymphoma zones. Lymphomas that occur after transplantation of bone marrow or stem cells are typically B-cell non-Hodgkin lymphomas.

Chronic lymphocytic leukemia (CLL) is a sluggish (slow-growing) cancer that causes a slow increase in the number of immature white blood cells called B-lymphocytes or B-cells. Cancer cells spread through the blood and bone marrow, and can also infect lymph nodes or other organs such as the liver and spleen. Ultimately, CLL causes bone marrow damage. Sometimes in the later stages of the disease, this disease is called small cell lymphocytic lymphoma.

In specific embodiments, methods are provided comprising administering a therapeutically effective amount of the CAR-expressing immune effector cells described herein or a composition containing them to a patient in need thereof, alone or in combination with one or more therapeutic agents. In certain embodiments, the cells of the present invention are used in the treatment of patients at risk of developing cancer. Thus, the present invention provides methods for treating or preventing cancer, comprising administering to a subject in need thereof a therapeutically effective amount of CAR-modified T cells of the present invention.

As used herein, the terms “individual” and “subject” are often used interchangeably and refer to any animal that has a cancer symptom that can be treated with gene therapy vectors, cell-based therapeutics, and methods disclosed elsewhere this document. Suitable subjects (e.g., patients) include laboratory animals (such as a mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients are included. Typical subjects are human patients who have cancer, who have been diagnosed with cancer, or who are at risk of developing cancer.

As used herein, the term “patient” refers to a subject who has been diagnosed with a specific cancer that can be treated with vectors for gene therapy, cell-based therapeutics, and methods disclosed elsewhere in this document.

As used herein, “treatment” or “treatment” includes any positive or necessary effect on the symptoms or pathological signs of a disease or condition, and may even include a slight decrease in one or more measurable markers of the disease or condition being treated, for example, cancer . Treatment may optionally involve either a reduction or amelioration of the symptoms of the disease or condition, or a delay in the progression of the disease or condition. "Treatment" does not necessarily mean the complete elimination or cure of the disease or condition, or related symptoms.

Used in this description, “warn” and similar words, such as “warned”, “warning”, etc., indicate an approach for preventing, inhibiting or reducing the likelihood of a disease or condition, for example, cancer. It also relates to delaying the manifestation or relapse of a disease or condition or delaying the onset or relapse of symptoms of a disease or condition. As used herein, “warning” and similar words also include a reduction in the intensity, effect, symptoms and / or burden of the disease or condition prior to the manifestation or relapse of the disease or condition.

“Strengthen,” or “promote,” or “increase,” or “enhance” generally refers to the ability of a composition described herein, for example, a genetically modified T cell or CAR encoding vector, to produce, promote, or induce a stronger physiological reaction (i.e. subsequent effects) as compared to a reaction caused by either a carrier or a control molecule / composition. The measured physiological response may include an increase in the multiplication, activation, persistence of T cells and / or an increase in their ability to cause cytolysis of cancer cells, among other things, obvious from the understanding in the prior art and the description herein. An “increased” or “increased” amount is typically a “statistically significant” amount, and may include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 15, 20, 30 or more times (for example, 500, 1000 times) (including all integers and decimal places between them, greater than 1, for example, 1.5, 1.6, 1.7 , 1.8, etc.) exceeds the reaction due to the carrier or control composition.

“Reduce,” or “lower,” or “lighten,” or “lower,” or “weaken” generally refers to the ability of the composition described herein to produce, promote, or induce a weaker physiological response (i.e., subsequent effects ) compared with a reaction caused by either a carrier or a control molecule / composition. A “reduced” or “reduced” amount is typically a “statistically significant” amount, and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 15, 20, 30 or more times (for example, 500, 1000 times) (including all integers and decimal places between them, greater than 1, for example, 1.5, 1.6, 1.7 , 1.8, etc.) is less in comparison with the reaction (reference reaction) caused by the carrier, the control composition, or the reaction in a particular cell line.

“Maintain” or “maintain” or “maintain” or “without change” or “without significant changes” or “without significant decrease” generally refers to the ability of the composition described in this document to produce, promote or cause more a weak physiological reaction (i.e. subsequent effects) in the cell compared to a reaction caused by either a carrier or a control molecule / composition, or a reaction in a particular cell line. A commensurate reaction is a reaction that is not substantially different or measurably different from a reference reaction.

In one embodiment, a method of treating cancer in a subject in need thereof, comprises administering an effective amount, for example, a therapeutically effective amount, of a composition comprising the genetically modified immune effector cells described herein. The amount and frequency of administration will be determined depending on factors such as the patient’s condition and the type and severity of the patient’s disease, although appropriate dosages can be determined using clinical trials.

In certain embodiments, it may be necessary to administer activated T cells to the subject, and then sequentially take blood (or perform apheresis), activate the T cells obtained from it in accordance with the present invention, and reinfuse these activated and multiplied T cells to a patient . This process can be carried out many times every few weeks. In certain embodiments, T cells can be activated from blood taken in an amount of 10 cm ³ to 400 cm ³ . In certain embodiments, T cells are activated from blood taken in an amount of 20 cm ³ , 30 cm ³ , 40 cm ³ , 50 cm ³ , 60 cm ³ , 70 cm ³ , 80 cm ³ , 90 cm ³ , 100 cm ³ , 150 cm ³ , 200 cm ³ , 250 cm ³ , 300 cm ³ , 350 cm ³ or 400 cm ³ or more. Without being attached to any theory, the use of such a multiple blood sampling / multiple reinfusion protocol can serve to select certain populations of T cells.

The introduction of the compositions discussed in this document can be carried out in any convenient way, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. In a preferred embodiment, the compositions are administered parenterally. As used herein, the phrases “parenteral administration” and “administered parenterally” refer to administration methods other than enteral and local administration, which are usually carried out by injection and which include, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraocular, intratumoral, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, in trapinal and intrathoracic injection and infusion. In one embodiment, the compositions described herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

In one embodiment, an effective amount of the composition is administered to a subject in need thereof to enhance the cellular immune response to cancer in the subject. The immune response may include cellular immune responses mediated by cytotoxic T cells capable of leading to cytolysis of infected cells, regulatory T cells, as well as reactions with helper T cells. Humoral immune responses can also be induced, primarily mediated by helper T cells capable of activating B cells, resulting in antibody production. A variety of techniques can be used to analyze the type of immune responses induced by the compositions of the present invention, which are well described in the art; e.g. Current Protocols in Immunology, ed. John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, N.Y.

In the case of T-cell-mediated cell cytolysis, the binding of a CAR ligand initiates signal transduction via CAR to the T cell, which leads to the activation of a number of signal transduction pathways in the T cell, which induces the T cell to produce or release proteins capable of inducing apoptosis of the target cell through various mechanisms. Such T-cell-mediated mechanisms include (without limitation) the transfer of intracellular cytotoxic granules from the T-cell to the target cell, T-cell secretion of anti-inflammatory cytokines, which can induce cell cytolysis directly (or indirectly by recruiting killer effector cells), and enhancing ligand expression death receptors (e.g., FasL) on the surface of T cells that induce apoptosis of the target cell after binding to its cognate death receptor (e.g., Fas) on the target glue kyo.

In one embodiment of the present invention, there is provided a method of treating a subject who has been diagnosed with cancer, comprising extracting the immune effector cells from the subject, genetically modifying said immune effector cells with a vector comprising the nucleic acid encoding the CAR discussed herein to obtain the same population of modified immune effector cells, and the introduction of modified immune effector cells to the same subject. In a preferred embodiment, the immune effector cells include T cells.

In certain embodiments of the present invention, methods are also provided for stimulating an immunomodulatory response mediated by immune effector cells against a population of target cells in a subject, comprising the steps of administering to the subject a population of immune effector cells expressing a nucleic acid construct encoding a CAR molecule.

Methods for administering the cell-based compositions described herein include any method that is effective either for re-administering ex vivo genetically modified immune effector cells that directly express the CAR of the present invention in a subject, or for re-administering genetically modified immune effector precursors cells that, when introduced into a subject, differentiate into mature immune effector cells expressing CAR. One method involves ex vivo transduction of peripheral blood T cells using the nucleic acid construct of the present invention and returning the transduced cells to the subject.

In certain embodiments, the folate receptor alpha, 5T4, α _v β ₆ integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a , CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, GD2, GD2, GD2 glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO-1, HLA-A3 + NY -ESO-1, IL-11Rα, IL-13Rα2, lambda chain, Lewis-Y, kappa chain, mesothelin, Muc1, Muc16, NCAM, ligands NKG2D, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGFR2 polynucleotides, polypeptides, fragments of polypeptides or antibodies to them are part of the concomitant diagnosis The rest of the method, usually used to assess whether a subject or a population of subjects will respond positively to a specific therapeutic treatment.

As used herein, the term “concomitant diagnostic” refers to a diagnostic test that is associated with a particular CAR or therapy with immune effector cells. In a specific embodiment, diagnostic methods and kits comprise determining expression levels of a folate receptor polypeptide or polynucleotide alpha, 5T4, α _v β ₆ integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphAAP, Ep , fetal AchR, FRα, GD2, GD3, 'glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO-1, HLA-A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chains, Lewis-Y, kappa chains, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY-ESO -1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGF R2 in a biological sample, thereby allowing rapid identification of patients suitable for treatment in accordance with the present invention.

For example, a given cancer therapeutic agent (eg, containing CAR or genetically modified immune effector cells expressing the CARs described herein) can be identified as appropriate for a subject or a specific population of subjects based on whether the subject (s) is characterized the presence of one or more selected biomarkers for a given disease or condition. Examples of biomarkers include serum / tissue markers, as well as markers that can be identified using medical imaging techniques. In certain embodiments, the polypeptide fragment (or its corresponding polynucleotide) of the folate receptor alpha, 5T4, α _v β ₆ integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6 , CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fet , FRα, GD2, GD3, 'glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO -1, HLA-A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chains, Lewis-Y, kappa chains, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGFR2 may themselves provide serum and / or a tissue biomarker that can be used to measure drug treatment results or to evaluate the feasibility of using a drug in a particular subject or a specific population of subjects. In certain aspects, identifying a treatable indication that expresses the polypeptide or corresponding polynucleotide sequence of the folate receptor alpha, 5T4, α _v β ₆ integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33 , CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR families, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, Ep FAP, Fetal AchR, FRα, GD2, GD3, Glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO-1, HLA-A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chains, Lewis-Y, kappa chains, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY- ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, urvivina, TAG72, TEM or VEGFR2 may include determining the differential expression of a sequence or the selected entity in the selected tissue, or otherwise as described herein and known in the art.

In a specific embodiment, the methods described herein include measuring or quantifying pre-mRNA, mRNA, or protein expression of a folate receptor polypeptide alpha, 5T4, α _v β ₆ integrin, BCMA, B7-H3, B7-H6, CAIX , CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family, including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, GD2, GD3, 'glypican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO-1, HLA-A3 + NY-ESO-1, IL-11Rα, IL-13Rα2, lambda chains, Lewis-Y, kappa chains, mesothelin, Muc1 , Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEM or VEGFR2 in a cancer of a subject. In one embodiment, a subject is identified as having a particular cancer treatable by the compositions described herein if the expression of the marker is 10 times, 25 times, 50 times, 100 times, or 1000 times higher or more in a biological sample than marker expression in a control sample or in a known standard. In a specific embodiment, the subject is identified as being treatable if the expression of the biomarker in the biological sample is detectable and the expression of the marker in a control sample or known standard using the same method below the detection level.

The presence, absence or relative levels of protein expression of a biomarker in a potential cancer can be analyzed using, for example, histochemical methods, immunological methods, electrophoresis, Western blot analysis, FACS analysis, flow cytometry, etc. In addition, the presence, absence or relative levels of biomarker RNA expression can be detected, for example, by PCR, Northern blot analysis, the use of suitable oligonucleotide probes, etc.

All publications, patent applications, and granted patents cited herein are incorporated herein by reference, as if each individual publication, patent application, or patent granted were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in sufficient detail for illustration and example for purposes of clarity of understanding, it will be apparent to one of ordinary skill in the art in light of the ideas of the present invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Specialists in this field can easily identify a number of secondary parameters that could be changed or modified to obtain essentially the same results.

EXAMPLES

EXAMPLE 1

CONSTRUCTION CAR

1. CD19-specific CAR (pMND-CD19 CAR)

CD19-specific CARs were developed containing an MND promoter operably linked to an anti-CD19 scFv antibody, a hinge and transmembrane domain from CD8α, and CD137 costimulation domains, behind which an intracellular signal transmission domain from the CD3ζ chain is located. Figure 1A. CD19-CAR contains a CD8α signal peptide (SP) sequence for expression on the surface of immune effector cells. The polynucleotide sequence of pMND-CD19-CAR is set forth under SEQ ID NO: 2 and the vector map is shown in Figure 2. Table 3 shows the designation, individual code in Genbank, source name and bibliographic reference for the various nucleotide segments of the lentiviral vector pMND-CD19-CAR

Table 3.

Nucleotides Designation Individual code in GenBank Source Name Bibliographic reference 1-185 The backbone of the plasmid pUC19 Access No. L09137.2
nt 1–185 pUC19 New england biolabs 185-222 Linker Not applicable Synthetic Not applicable 223-800 CMV Not applicable pHCMV (1994) PNAS 91: 9564-68 801-1136 R, U5, PBS and packaging sequences Access No. M19921.2
nt 454-789 pNL4-3 Maldarelli, et.al. (1991)
J Virol: 65 (11): 5732-43 1137-1139 Gag start codon (ATG) changed to stop codon (TAG) Not applicable Synthetic Not applicable 1140-1240 The gag sequence of HIV-1 Access No. M19921.2
nt 793-893 pNL4-3 Maldarelli, et.al. (1991)
J Virol: 65 (11): 5732-43 1241-1243 HIV-1 gag sequence changed to second stop codon Not applicable Synthetic Not applicable 1244-1595 The gag sequence of HIV-1 Access No. M19921.2
nt 897-1248 pNL4-3 Maldarelli, et.al. (1991)
J Virol: 65 (11): 5732-43 1596-1992 Pol HIV-1
cPPT / CTS Access No. M19921.2
nt 4745-5125 pNL4-3 Maldarelli, et.al. (1991)
J Virol: 65 (11): 5732-43 1993-2517 HIV-1, env region (RRE) of HXB3 isolate Access No. M14100.1
nt 1875-2399 PgTAT-CMV Malim, MH
Nature (1988) 335: 181-183 2518-2693 The env sequences of HIV-1 S / A Access No. M19921.2
nt 8290-8470 pNL4-3 Maldarelli, et.al. (1991)
J Virol: 65 (11): 5732-43 2694-3231 Mnd Not applicable pccl-c-MNDU3c-x2 Challita et al. (1995)
J. Virol. 69: 748-755 3232-3247 Linker Not applicable Synthetic Not applicable 3248-3310 Signal peptide Access Number NM_001768 Synthetic Not applicable 3311-4036 scFv to CD19 (FMC63) Not applicable Synthetic Not applicable 4037-4243 Hinge area and TM CD8a Access Number NM_001768 Synthetic Milone et al (2009)
Mol Ther 17 (8): 1453-64 4244-4369 Signal Transmission Domain CD137 (4-1BB) Access Number NM_001561 Synthetic Milone et al (2009)
Mol Ther 17 (8): 1453-64 4370-4708 CD3-ζ Signal Transfer Domain Access Number NM_000734 Synthetic Milone et al (2009)
Mol Ther 17 (8): 1453-64 4709-4838 ppt and part 3 ’U3 HIV-1 Access No. M19921.2
NT 9005-9110 pNL4-3 Maldarelli, et.al. (1991)
J Virol: 65 (11): 5732-43 4839-4935 R and part 3 ’U3 (399 bp removed in U3) HIV-1 Access No. M19921.2
nt 9511-9627 pNL4-3 Maldarelli, et.al. (1991)
J Virol: 65 (11): 5732-43 4936-4961 Synthetic polyA Not applicable Synthetic Levitt, N. Genes & Dev (1989) 3: 1019-1025 4962-5010 Linker Not applicable Synthetic Not applicable 5011-7425 Skeleton pUC19 Access No. L09137.2
nt 2636-2686 pUC19 New england biolabs

2. CAR specific for light gear chain ( _LC gear ₎ (pMND-kappa-CAR)

Developed are CARs specific for a light chain, containing an MND promoter operably linked to a scFv antibody to a light chain, a hinge and transmembrane domain from CD8α and CD137 costimulation domains, behind which there is an intracellular signal transmission domain from the CD3ζ chain . Figure 1B. Kappa _LC- CAR contains a CD8α signal peptide (SP) sequence for expression on the surface of immune effector cells. The polynucleotide sequence of the pMND-kappa _LC- CAR is set forth under SEQ ID NO: 3 and the vector map is shown in Figure 3. Table 4 shows the designation, individual code in Genbank, source name and bibliographic reference for the different nucleotide segments of the lentiviral vector pMND-light kappa- chain-CAR.

Table 4.

Nucleotides Designation Individual code in GenBank Source Name Bibliographic reference 1-185 The backbone of the plasmid pUC19 Access No. L09137.2
nt 1–185 pUC19 New england biolabs 185-222 Linker Not applicable Synthetic Not applicable 223-800 CMV Not applicable pHCMV (1994) PNAS 91: 9564-68 801-1136 R, U5, PBS and packaging sequences Access No. M19921.2
nt 454-789 pNL4-3 Maldarelli, et.al. (1991)
J Virol: 65 (11): 5732-43 1137-1139 Gag start codon (ATG) changed to stop codon (TAG) Not applicable Synthetic Not applicable 1140-1240 The gag sequence of HIV-1 Access No. M19921.2
nt 793-893 pNL4-3 Maldarelli, et.al. (1991)
J Virol: 65 (11): 5732-43 1241-1243 HIV-1 gag sequence changed to second stop codon Not applicable Synthetic Not applicable 1244-1595 The gag sequence of HIV-1 Access No. M19921.2
nt 897-1248 pNL4-3 Maldarelli, et.al. (1991)
J Virol: 65 (11): 5732-43 1596-1992 Pol HIV-1
cPPT / CTS Access No. M19921.2
nt 4745-5125 pNL4-3 Maldarelli, et.al. (1991)
J Virol: 65 (11): 5732-43 1993-2517 HIV-1, env region (RRE) of HXB3 isolate Access No. M14100.1
nt 1875-2399 PgTAT-CMV Malim, MH
Nature (1988) 335: 181-183 2518-2693 The env sequences of HIV-1 S / A Access No. M19921.2
nt 8290-8470 pNL4-3 Maldarelli, et.al. (1991)
J Virol: 65 (11): 5732-43 2694-3231 Mnd Not applicable pccl-c-MNDU3c-x2 Challita et al. (1995)
J. Virol. 69: 748-755 3232-3245 Linker Not applicable Synthetic Not applicable 3246-3302 Signal peptide Synthetic Not applicable 3303-4061 scFv to kappa chain Not applicable Synthetic Not applicable 4062-4268 Hinge area and TM CD8a Access Number NM_001768 Synthetic Milone et al (2009)
Mol Ther 17 (8): 1453-64 4269-4394 Signal Transmission Domain CD137 (4-1BB) Access Number NM_001561 Synthetic Milone et al (2009)
Mol Ther 17 (8): 1453-64 4395-4733 CD3-ζ Signal Transfer Domain Access Number NM_000734 Synthetic Milone et al (2009)
Mol Ther 17 (8): 1453-64 4734-4960 ppt, U3 and R HIV-1 Access No. M19921.2
NT 9005-9110 pNL4-3 Maldarelli, et.al. (1991)
J Virol: 65 (11): 5732-43 4961-4985 Synthetic polyA Not applicable Synthetic Levitt, N. Genes & Dev (1989) 3: 1019-1025 4986-5025 Linker Not applicable Synthetic Not applicable 5026-7450 Skeleton pUC19 Access No. L09137.2
nt 2636-2686 pUC19 New england biolabs

EXAMPLE 2

T-CELL TRANSDUCTION

Lentiviral vector (LV) supernatants were obtained in HEK 293T cells as described in the literature (Naldini et al., 1996, Dull et al., 1998 and Zufferey et al., 1998). Transient transfection with 5 plasmids (HPV 275 encoding HIV gag-pol, ψN 15 encoding VSV-G envelope protein, p633 encoding HIV rev protein, HPV601 encoding HIV tat protein and CAR expression vector) was used as described in the publication according to PCT No. WO2012 / 170911. Then, LV supernatants were concentrated either by ultracentrifugation or by means of an ion exchange column followed by tangential flow filtration (TFF), prepared in SCGM medium (CellGenix Inc., Germany) and cryopreserved at <-70ºC in single-use cryophials. Infectious titers were determined using flow cytometry analysis of transduced human osteosarcoma cells (HOS) (Kutner et al., 2009, Nature Protocols 4: 495-505). For transduction of human T lymphocytes, primary human T cells were isolated from the blood of healthy volunteer donors after leukapheresis by negative selection using RosetteSep kits (Stem Cell Technologies). T cells were cultured in RPMI 1640 supplemented with 10% FCS, 100 U / ml penicillin, 100 g / ml streptomycin sulfate, 10 mM Hepes, and stimulated with magnetic beads coated with anti-CD3 / anti-CD28 antibody in a cell to pellet ratio 1: 3. In the case of CD8 T cells, human IL-2 (Chiron) was added every other day to a final concentration of 30 IU / ml. About 24 hours after activation, T cells were transduced using lentiviral vectors at an MOI of 5. The transduction of T cells was evaluated by polymerase chain reaction using primers specific for the viral vector and flow cytometry after 7-10 days after transduction.

EXAMPLE 3

VCN IN T-CELLS TRANSDUCED WITH CAR

The number of copies of the vector was determined in the case of transduction of primary human T cells using lentivirus pMND-kappa _LC- CAR. Peripheral blood mononuclear cells (PBMCs) were collected from normal donors and activated by culturing with antibodies specific for CD3 and CD28 (Miltenyi Biotec) in a medium containing IL-2 (CellGenix). After culture activation, PBMCs were transduced using lentiviral vectors or left untreated. Cultures were maintained to ensure the growth and reproduction of T cells (7-10 days). At the time of harvesting the culture contained T cells, which multiplied to a level of about 2 log.

The number of copies of the vector (VCN) of the integrated lentiviral particles was determined by quantitative PCR nine after transduction. The average VCN for 12 unique cultures from 6 donors was 3.1. Figure 4.

EXAMPLE 4

CAR EXPRESSION IN TRANSDUCED T-CELLS

The expression of chimeric antigenic receptors specific for the kappa chain expressed by the MND promoter (pMND-kappa _LC -CAR) on the cell surface of primary human T cells was determined. Peripheral blood mononuclear cells (PBMCs) were collected from normal donors and activated by culturing with antibodies specific for CD3 and CD28 (Miltenyi Biotec) in a medium containing IL-2 (CellGenix). After culture activation, PBMCs were transduced using lentiviral vectors or left untreated. Cultures were maintained to ensure the growth and reproduction of T cells (7-10 days). At the time of harvesting the culture contained T cells, which multiplied to a level of about 2 log.

Kappa _LC expression was determined by flow cytometry using mouse Ig specific antibodies (BD Biosciences), which are present only on T cells modified with pMND Kappa _LC CAR. Flow cytometry was performed 6–9 days after transduction. The average expression level of Kappa _LC in 12 unique cultures from 6 donors was 35.6%. Figure 5.

EXAMPLE 5

MND PROMOTOR PROVIDES CAR EXPRESSION IN T-CELLS AT LEVEL COMPARED TO SUCH PROVIDED BY EF1Α PROMOTOR

The expression of CD19-CAR on modified T cells provided by the MND promoter was comparable to the expression of CD19-CAR provided by the EF1α promoter. Peripheral blood mononuclear cells (PBMCs) were collected from normal donors and activated by culturing with antibodies specific for CD3 and CD28 (Miltenyi Biotec) in a medium containing IL-2 (CellGenix). After culture activation, PBMCs were transduced using lentiviral vectors or left untreated. Cultures were maintained to ensure the growth and reproduction of T cells (7-10 days). At the time of harvesting the culture contained T cells, which multiplied to a level of about 2 log. At the end of the cultivation, T-cell transduction was evaluated by quantitative polymerase chain reaction (qPCR) using primers specific for viral particles. CD19-CAR expression was determined six days after transduction by flow cytometry using murine Ig specific antibodies (BD Biosciences), which are present only on T cells modified with CD19 CAR. Both CD19-CAR and VCN expression were comparable for different constructs. Figure 6.

EXAMPLE 6

CAR-T CELL ANTIGEN-SPECIFIC REACTIVITY

The antigen-specific reactivity of T cells with pMND-kappa _LC- CAR was determined. Peripheral blood mononuclear cells (PBMCs) were collected from normal donors and activated by culturing with antibodies specific for CD3 and CD28 (Miltenyi Biotec) in a medium containing IL-2 (CellGenix). After culture activation, PBMCs were transduced using lentiviral vectors or left untreated. Cultures were maintained to ensure the growth and reproduction of T cells (7-10 days). At the time of harvesting the culture contained T cells, which multiplied to a level of about 2 log.

At the end of the cultivation, tumor reactivity was evaluated by the release of interferon-gamma (IFNγ). T cells modified with pMND-kappa _LC- CAR secrete IFNγ after co-culture with kappa ⁺ Daudi cells (express kappa _LC ). In contrast, co-culture of T cells modified with pMND-kappa _LC- CAR with kappa-negative HDLM-2 cells resulted in the release of IFNγ in an amount comparable to that of T-cell culture alone. IFNγ release was determined using ELISA kits 24 hours after co-culture with kappa-positive Daudi cells or kappa-negative HDLM-2 cells. Figure 7.

EXAMPLE 7

CAR-T-CELL ANTITUM FUNCTION

The antitumor function of CAR-T cells engineered to express pMND kappa _LC- CAR was determined. Peripheral blood mononuclear cells (PBMCs) were collected from normal donors and activated by culturing with antibodies specific for CD3 and CD28 (Miltenyi Biotec) in a medium containing IL-2 (CellGenix). After culture activation, PBMCs were transduced using lentiviral vectors or left untreated. Cultures were maintained to ensure the growth and reproduction of T cells (7-10 days). At the time of harvesting, the cells contained T cells that multiplied to a level of about 2 log.

2 x 10 ⁶ Daudi cells labeled with the firefly luciferase gene were implanted into IL-2 receptor gamma chain knockout NOD scam mice via intravenous injection. After three, six, and nine days after injection of tumor cells into mice 1x10 ⁷ T cells modified with MND-kappa _LC -CAR, adoptively transferred into mice and tumor growth was monitored using bioluminescent imaging system using Xenogen-IVIS. Tumor burden in mice injected with modified CAR-T cells decreased compared with tumor burden in untreated mice. Figure 8.

EXAMPLE 8

OBTAINING A FUNCTIONAL MEDICINE CONTAINING CAR-T CELLS

T cells expressing CAR for BCMA were obtained as described in Example 1 above. Such CAR-T cells showed antigen-specific tumor clearance. T cells expressing CAR for BCMA were co-cultured for 4 hours with K562 cells or K562 cells modified for BCMA expression. Tumor cells expressing the antigen were labeled with carboxyfluorescein (CFSE) succinimidyl ester and fluorescence was measured using FACS. T cells expressing CAR for BCMA led to the cytolysis of K562 cells expressing BCMA (Figure 9A) and IFN-γ was released (Figure 9B). (n = 3).

In general, in the following claims, the terms used should not be construed as limiting the claims to the specific embodiments disclosed in the present description and claims, but should be construed as including all possible embodiments together with the full scope of equivalents that such claims cover. Accordingly, the claims are not limited to the present disclosure.

Claims (21)

1. Lentiviral vector for the treatment of cancer, containing the left (5 ') lentiviral LTR, containing the heterologous cytomegalovirus (CMV) promoter, Psi (Ψ) packaging signal, central polypurine pathway / DNA flap (cPPT / FLAP), rev-response element ( RRE); (3 ') lentiviral self-inactivating (SIN) LTR; synthetic polyadenylation sequence; and a polynucleotide containing a promoter (MND) with an enhancer of the myeloproliferative sarcoma virus, with a deleted negative control region, with a primer binding site replaced by a sequence from dl587rev, operably linked to a nucleic acid encoding a chimeric antigenic receptor (CAR), wherein CAR contains: (a) scFv, which binds the B-cell maturation antigen (BCMA); (b) the hinge region of CD8α; (c) a transmembrane domain from CD8α; (d) CD137 costimulatory signal transmission domain; and (e) CD3ζ primary signal transmission domain. 2. The lentiviral vector of claim 1, wherein the scFv is human, murine, or humanized. 3. The lentiviral vector of claim 1 or 2, wherein the CAR further comprises a hinge region polypeptide, a spacer region, or a signal peptide. 4. The lentiviral vector according to claim 1, wherein the lentiviral vector is selected from the group consisting of: human immunodeficiency virus (HIV); Visna Maedi virus (VMV); goat arthritis encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV) and monkey immunodeficiency virus (SIV). 5. Lentiviral vector according to claim 1, additionally containing: a) a heterologous polyadenylation sequence; b) a heterologous polyadenylation sequence, which is a bovine growth hormone polyadenylation sequence or rabbit β-globin polyadenylation signal sequence; or c) hepatitis B virus post-transcriptional regulatory element (HPRE) or marmot hepatitis virus post-transcriptional regulatory element (WPRE). 6. T-lymphocyte for the treatment of cancer, containing the lentiviral vector according to claim 1. 7. A composition for the treatment of cancer containing an effective amount of T-lymphocytes according to claim 6 and a physiologically acceptable excipient. 8. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition according to claim 7. 9. The method of claim 8, wherein the cancer is hemoblastosis. 10. The method of claim 9, wherein the hemoblastosis is a B-cell neoplasm selected from the group consisting of multiple myeloma (MM), chronic lymphocytic leukemia (CLL), or non-Hodgkin's lymphoma (NHL). 11. The method of claim 10, wherein the MM is selected from the group consisting of clinically severe multiple myeloma, sluggish multiple myeloma, plasmacytic leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma and extramedullary plasmacytoma. 12. The method of claim 10, wherein the NHL is selected from the group consisting of Burkitt’s lymphoma, chronic lymphocytic leukemia / small cell lymphocytic lymphoma (CLL / SLL), diffuse B-large cell lymphoma, follicular lymphoma, immunoblastic large-cell lymphoma, lymphoblastic lymphoma from B-cells precursors and mantle cell lymphoma.

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